Monomeric recombinant mhc molecules useful for manipulation of antigen-specific t-cells

ABSTRACT

The present invention provides, in particular embodiments, for modified recombinant T cell receptor (TCR) ligands (RTLs) comprising a MHC class I or MHC class II component. The modified RTLs have redesigned surface features that preclude or reduce aggregation, wherein the modified molecules retain the ability to bind Ag-peptides, target antigen-specific T cells, inhibit T cell proliferation in an Ag-specific manner and have utility to treat, inter alia, autoimmune disease and other conditions mediated by antigen-specific T cells in vivo.

CROSS REFERENCES TO RELATED APPLICATIONS

The present application claims the priority benefit of U.S. ProvisionalPatent Application No. 60/500,660, filed by Burrows et al. on Sep. 5,2003, which is incorporated herein by reference

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

Aspects of this work were supported by grants from the NationalInstitutes of Health (A143960, ESI0554 and NS41965), the NationalMultiple Sclerosis Society (RG3012A), and the Department of VeteransAffairs. The United States government has certain rights in the subjectmatter.

FIELD OF THE INVENTION

The present invention relates to recombinant polypeptides comprisingmajor histocompatibility complex (MHC) molecular domains that mediateantigen binding and T cell receptor (TCR) recognition, and to relatedcompositions and methods incorporating these recombinant polypeptides.The compositions and methods of the invention are useful for detection,quantification, and purification of antigen-specific T cells, formodulating T cell activity, and for treating T cell mediated diseasessuch as autoimmune disorders.

BACKGROUND OF THE INVENTION

The immune system ordinarily functions to direct protective immuneresponses against microorganisms and other harmful foreign materials. Inthe context of autoimmune diseases and transplant rejection, however,these normally beneficial immune responses can mediate deleterious andoften fatal effects. In the case of autoimmunity, antigens present inthe body's own tissues become targets for autoreactive immune responsesthat cause tissue destruction and other disease symptoms.

Immune responses in mammals are mediated by a diverse array ofperipheral blood cells called leukocytes. Leukocytes arise fromhematopoietic stem cells which undergo self-renewal and differentiationinto two precursor lineages—the myeloid and lymphoid lines. Furtherdifferentiation occurs among these lineages to produce monocyte,eosinophil, neutrophil, basophil, megakaryocyte, and erythroid cellsfrom the myeloid line, and T lymphocytes, B lymphocytes, and NK cellsfrom the lymphoid line.

T lymphocytes include CD8+ T cells (cytotoxic/suppressor T cells), andCD4+ T cells distinguished in part by their expression of cell surfacemolecules, CD8, and CD4, respectively, which function to enhance theavidity with which T cells bind antigen-bearing or target cells, and mayalso promote the interaction of the TCR with cognate antigen. Bierer etal., Ann. Rev. Immunol. 7:579-99, 1989.

CD4+ T cells play a key regulatory role with respect to other immunesystem cell types, acting as “T helper” or “T inducer” cells whenactivated. By virtue of this central regulatory role, CD4⁺ T cells arekey players in the pathogenesis of various autoimmune diseases,including multiple sclerosis (MS), rheumatoid arthritis (RA), diabetes,sarcoidosis, autoimmune uveitis, chronic beryllium disease, and are alsoconsidered to play a causal role in transplant rejection andgraft-versus-host disease (GVHD) (Swanborg, J. Immunol 130:1503-05,1983; Cush. Arthritis Rheum. 31: 1230-38, 1988; Caspi, J. Immunol140:1490-95, 1988; Cobbold et al., Nature 312:54851, 1988; Steinman,Sci. Am. 269:106-14, 1993).

CD4⁺ T cells mediate their role in autoimmune disease by responding inan antigen-specific manner to “autoantigens” associated with targetcells or tissues. Pathogenic CD4+ T cells migrate or “home” to targettissues hearing autoantigen and selectively produce T-helper type 1(Th1) cytokines, which trigger recruitment and activation of otherlymphocytes and monocytes that may destroy target tissues and causeother adverse disease sequelae (Weinberg, et al., J. Immunol148:2109-17, 1992; Weinberg et al., J. Immunol 152:4712-5721, 1994).

Normal activation of T lymphocytes occurs when the T cells interact withantigen-presenting cells (APCs) bearing cognate antigen (Ag) in thecontext of a major histocompatibility complex (MHC) protein. Thespecificity of T cell responses is conferred by a polymorphic,antigen-specific T cell receptor (TCR). T cell activation is mediated byTCR recognition of the Ag presented on the surface of the APC as aprocessed peptide bound to the MHC molecule.

Two distinct classes of MHC molecules occur in humans and other mammals,termed MHC class I and MHC class II. Both classes of MHC moleculescomprise complexes formed by association of multiple polypeptide chains,and each includes a trans-membrane portion that anchor the complex intothe APC membrane. MHC class I molecules are comprised of anα-polypeptide chain non-covalently associated with a β2-microglobulinchain. The α-chain of MHC class I includes three distinct domains,termed the α1, α2 and α3 domains. The three-dimensional structure of theα1 and α2 domains of MHC I molecules forms a peptide binding groove(alternatively referred to herein as the peptide binding cleft orpocket) which binds cognate Ag for presentation to T-cells. The αβdomain is an Ig-fold like domain that includes a trans-membrane sequenceto anchor the α-chain into the cell membrane of the APC. MHC class Icomplexes, when associated with antigen in the presence of appropriateco-stimulatory signals, stimulate CD8+ cytotoxic T-cells to kill targetcells in an Ag-specific manner.

The genes that encode the various polypeptide chains that associate toform MHC complexes in mammals have been studied and described inextensive detail. In humans, MHC molecules (with the exception of classI β2-microglobulin) are encoded in the HLA region of the genome, locatedon chromosome 6. There are three class I MHC α-chain-encoding loci,termed HLA-A, HLA-B and HLA-C. In the case of MHC class II proteins,there are three pairs of α and β chain loci, termed HLA-DR(A and B),HLA-DP(A and B), and HLA-DQ(A and B). In rats, the class I α gene isdesignated RT1.A, while the class H genes are termed RT1.B α and RT1.Bβ. More detailed description regarding the structure, function andgenetics of MHC complexes can be found, for example, in Immunobiology:The Immune System in Health and Disease by Janeway and Travers, CurrentBiology Ltd./Garland Publishing, Inc. (1997), and in Bodmer et al.(1994) “Nomenclature for factors of the HLA system” Tissue Antigens vol.44, pages 1-18.

The specificity of T cell responses is conferred by a polymorphic,antigen-specific T cell receptor (TCR). TCRs comprise multi-chain, α/βheterodimeric receptors, which are activated in an Ag-specific manner byAg processed and presented on the surface of APCs as a peptide bound tothe MHC complex. X-ray crystallographic data demonstrate that peptidesfrom processed antigen bind to MHC II proteins in a membrane distalpocket formed by the β1 and α1 domains (Matsui et al., Science254:1788-91, 1991; Nag et al., J. Biol. Chem. 267:22624-29, 1992).

CD4⁺ T cell activation generally follows a multi-step course thatincludes co-ligation of the TCR and CD4 by the MHC class 11/peptidecomplex presented by APCs. A separate activation event referred to as“co-stimulation” is mediated by other T cell surface molecules, such asCD28. In the absence of the second, co-stimulatory signal, stimulationof T cells through the TCR by MHC class II/peptide complex reportedlyinduces a state of unresponsiveness to subsequent optimal antigenpresentation, commonly referred to as “anergy”. (Quill, J. Immunol138:3704-12, 1987; Schwartz, J. Exp. Med. 184:1-8, 1996). In otherstudies, ligation of the TCR in the absence of a costimulatory signalhas been reported to disrupt normal T cell activation, inducing a rangeof responses from anergy to apoptosis (Schwartz, J. Exp. Med. 184:1-8,1996; Janeway, Cell 76:275-85, 1994; Burrows et al., J. Immunol167:4386-95, 2001; Wang et al., The Journal of Immunology, 2003).

MHC-restricted T lymphocyte interactions have been widely andextensively investigated. Cells of the T helper/inducer subset generallyrecognize antigen on the surface of APCs only in association with classII MHC gene products, which results in genetic restriction of antigenrecognition. While the rules governing the activation of MHC-restrictedT cells, and particularly of class II MHC-restricted T cells, have beenwell described, the underlying mechanisms are still being defined.

Despite the very large number of possible TCR specificities of T cells,a number of studies have shown that the major portion of the T cellresponse to protein antigens may be directed to a few “immunodominant”epitopes within the antigenic protein. In the context of autoimmunediseases, class II MHC-restricted T cell responses, and in some casesclinical signs of autoimmune disease, have been demonstrated to beassociated with specific proteins and/or immunodominant epitopes fromthese proteins, including, e.g., type II collagen (Rosloneic et al., J.Immunol. 160:2573-78, 1998; Andersson et al., Proc. Natl. Acad. Sci. USA95:7574-79, 1998; and Fugger et al., Eur. J. Immunol, 26:928-33, 1996),and human cartilage Ag gp39 (Cope et al., Arthritis Rheum. 42:1497,1999) associated with rheumatoid arthritis (RA), glutamic aciddecarboxylase 65 (Patel et al., Proc. Natl. Acad. Sci. USA 94:8082-87,1997; Wicker et al., J. Clin. Invest. 98:2597, 1996) and insulin (Congiaet al., Proc. Natl. Acad. Sci. USA 95:3833-38, 1998) associated withType 1 diabetes (insulin dependent diabetes mellitus or IDDM), andmyelin oligodendrocyte glycoprotein (MOG) (Forsthuber et al., J.Immunol. 167:7119, 2001) associated with MS and an animal disease modelfor MS, experimental autoimmune encephalomyelitis (EAE). Similarfindings have been reported for class II MHC-restricted T cell responsesassociated with myelin basic protein (MBP) (Madsen et al., Nat. Genet,23:343, 1999), proteolipid protein (PLP) (Kawamura et al., J. Clin.Invest, 105:977, 2000), and MOG (Vandenbark et al., J. Immunol.171:127-33, 2003).

One approach for managing and treating autoimmune diseases and other Tcell-mediated immune disorders is to regulate T cell activity usingnatural or synthetic TCR ligands, or T cell modulatory drugs or othercompounds, that are TCR agonists or antagonists. Various analogs ofnatural TCR ligands have been produced which comprise extracellulardomains of class II MHC molecules bound to a specific peptide Ag.Several such constructs have been purified as detergent extracts oflymphocyte membranes or produced as recombinant proteins (Sharma et al.,PNAS. 88:11465-69, 1991), Kozono et al., Nature 369:151-54, 1994;Arimilli et al., J. Biol. Chem. 270:971-77, 1995; Nag, PNAS 90:1604.08,1993; Nag et al., J. Biol. Chem. 271:10413-18, 1996; Rhode et al., J.Immunol. 157:4885-91, 1996; Fremont et al., Science 272:1001, 1996;Sharma et al., Proc. Natl. Acad. Sci. USA 88:11405, 1991; Nicolle etal., J. Clin. Invest. 93:1361, 1994; Spack et al., CNS Drug Rev. 4: 225,1998).

These two-chain, four-domain molecular complexes loaded with, orcovalently bound to, peptide Ag have been reported to interact with Tcells and modulate T cell activity in an Ag-specific manner (Matsui etal., Science 254:1788-91, 1991; Nag et al., J. Biol. Chem, 267:22624-29,1992; Nag, J. Biol. Chem. 268:14360-14366, 1993; Nag, PNAS 90:1604-08,1993; Nicolle et al., J. Clin. Invest. 93:1361-1369, 1994; Spack et al.,J. Autoimmun. 8:787-807, 1995). Various models have been presented forhow these complexes may be useful to modulate immune responses in thecontext of autoimmune disease. For example, U.S. Pat. No. 5,194,425(Sharma et al.) and U.S. Pat. No. 5,284,935 (Clark et al.) report theuse of isolated MHC class II/peptide complexes conjugated to a toxin toeliminate autoreactive T-cells. Others have reported the use of MHCII/antigen complexes, in the absence of co-stimulatory factors, toinduce a state of non-responsiveness in Ag-specific T cells known as“anergy” (Quill et al., J. Immunol., 138:3704-3712 (1987). Followingthis observation, Sharma et al. (U.S. Pat. Nos. 5,468,481 and 5,130,297)and Clarke et al. (U.S. Pat. No. 5,260,422) suggested that soluble MHCII/antigen complexes can be administered therapeutically to anergizeT-cell lines that specifically respond to autoantigenic peptides.Additional studies report that soluble MHC II/antigen complexes caninhibit 1′ cell activation, induce T cell anergy, and/or alleviate Tcell-mediated symptoms of autoimmune disease (Sharma et al., Proc. Natl.Acad. Sci. USA 88:11405, 1991; Spack et al., CNS Drub Rev. 4: 225, 1998;Steward et al., J. Allerg. Clin. Immun. 2:S117, 1997). In some cases, inthe absence of co-stimulation, intact MHC class II/peptide complexeshave been reported to modulate T cell activity by inducingantigen-specific apoptosis rather than anergy (Nag et al., J. Biol.Chem. 271:10413-18, 1996).

Although the concept of using isolated MHC/antigen complexes intherapeutic and diagnostic applications holds great promise, a majordrawback to the various methods reported to date is that the complexesare large and consequently difficult to produce and work with. Whilethese four domain complexes can be isolated from lymphocytes bydetergent extraction, such procedures are inefficient and yield onlysmall amounts of protein. Although cloning of genes encoding MHC complexsubunits has facilitated production of large quantities of individualsubunits through expression in prokaryotic cells, the assembly ofindividual subunits into MHC complexes having appropriate conformationalstructure has proven difficult. Another important feature of thesepreviously described, MHC II/antigen complexes is that they bind notonly to the TCR, but also to the CD4 molecule on the T cell surfacethrough the β2 MHC domain (Brogdon et al., J. Immunol. 161:5472, 1998).This additional interaction during peptide presentation and TCRengagement complicates the usefulness of prior MHC II/antigen complexesfor certain diagnostic and therapeutic applications. In addition,because of their size and complex structure, prior class II MHCcomplexes present an inherently difficult in vitro folding challenge.

To overcome these obstacles and provide additional advantages, inventorsin the current application previously developed novel, recombinant TCRligands or “RTLs” for use in modulating T cell activity. These RTLsincorporate selected structural components of a native MHC class IIprotein, typically comprising MHC class II α1 and β1 domains (orportions of the α1and β1 domains necessary to form a minimal, Ag-bindingpocket/TCR interface). These RTLs may exclude all or part of the β2domain of the MHC class II protein, typically at least the CD4-bindingportion of the β2 domain. Likewise, RTLs for use within the inventionmay exclude the α2 domain of the MHC class II protein (see, e.g.,Burrows et al., Prot. Eng. 12:771, 1999). Various RTLs having thesegeneral structural characteristics been produced in E. coli, with andwithout amino-terminal extensions comprising covalently bound, peptideAg.

These kinds of RTL constructs have been demonstrated to be effectiveagents for alleviating symptoms of CD4 + T cell-mediated autoimmunedisease in an MHC-specific, Ag-specific manner (Burrows et al., J.Immunol 167:4386-95. 2001; Vandenbark et al., Journal of immunology,2003). For example, RTL constructs have been tested and shown to preventand/or treat MBP-induced EAE in Lewis rats (Burrows et al., J. Immunol.161:5987, 1998; Burrows et al., J. Immunol. 164:6366, 2000) and toinhibit activation and induce IL-10 secretion in human DR2-restricted Tcell clones specific for MBP-85-95 or BCR-ABL b3a2 peptide (CABL)(Burrows et al., J. Immunol. 167:4386, 2001; Chang et al., J. Biol.Chem. 276:24170, 2001). Another RTL construct designed by inventors inthe current application is a MOG-35-55/DR2 construct (VG312) thatpotently inhibits autoimmune responses and elicits immunologicaltolerance to encephalitogenic MOG-35-55 peptide, and alleviates orreverses clinical and histological signs of EAE (Ivrandenbark et al., J.Immunol. 171:127-33, 2003). Numerous additional RTL constructs usefulfor modulating T cell immune responses have been developed by thecurrent inventors, which can be effectively employed within thecompositions and methods of the instant invention (see, e.g., Huan etal., J. Immunol. 172:4556-4566, 2004).

In recently described protein engineering studies of RTLs, applicantsdiscovered that MEIC class II-derived RTL molecules can foam undesirableaggregates in solution. In the case of one RTL construct derived fromHLA-DR2 (DRB1*1501/DRA*0101)), the purified RTL yielded approximately10% of the molecules in the form of stable dimers, with a remainingpercentage of the molecules found in the form of higher-order structuresabove 300,000 Daltons (Chang et al., J. Biol. Chem. 276:24170-76, 2001).

Although RTL aggregates retain biological activity (Burrows et al., J.Immunol 167:4386-95, 2001; Vandenbark et al., Journal of Immunology171:127-133, 2003), conversion of multimeric RTLs into a monodispersereagents in solution remains an important, unfulfilled objective tofacilitate use of RTLs as human therapeutics, for example to treatmultiple sclerosis and other autoimmune conditions.

Accordingly, there remains an unmet need in the art to providerecombinant TCR ligands (RTLs) that retain the ability to bind Agpeptides and interface functionally with a TCR to modulate T cellactivity in an Ag-specific manner, which have diagnostic and/ortherapeutic utility, and which exhibit a reduced potential foraggregation in solution or following administration to a mammaliansubject.

SUMMARY OF THE INVENTION

The present invention satisfies this need and fulfills additionalobjects and advantages by providing modified, recombinant T cellreceptor ligands (RTLs) that have been structurally modified to exhibita diminished propensity for self-aggregation. Modified RTLs of theinvention typically have one or more redesigned surface structuralfeatures introduced into an otherwise native MHC polypeptide sequence.For example, modified RTLs can be rationally designed and engineered tointroduce one or more amino acid changes at a solvent-exposed targetsite for modification located within, or defining, a self-binding (orself-associating) interface found in the native MHC polypeptide.

Within exemplary embodiments of the invention, the modified RTL includesa multi-domain structure comprising multiple MHC class I or MHC class IIdomains, or portions thereof necessary to form a minimal TCR interfacenecessary to mediate Ag binding and TCR recognition.

In the case of modified RTLs derived from human class II MHC molecules,the RTLs typically comprise α1 and β1 MHC polypeptide domains (orportions thereof sufficient to provide a minimal TCR interface) of anMHC class II protein. These domains or subportions thereof may becovalently linked to form a single chain (sc) MHC class II polypeptide.The resulting MHC component may be useful as an “empty” RTL, or may beassociated with a peptide Ag.

Modified RTL molecules of the invention show improved characteristics ofmonodispersal in aqueous solutions, while retaining their ability tobind peptide Ags, to target and modulate activity of antigen-specific Tcells, and to treat, inter cilia, autoimmune diseases and otherconditions mediated by antigen-specific T cells in vivo.

The modified RTLs of the invention lack certain structural featuresfound in intact, native MHC molecules (e.g., trans-membrane Ig folddomains), but nonetheless are capable of refolding in a manner that isstructurally analogous to native whole MHC molecules. The modified RTLsare likewise capable of binding peptide Ags to form stable MHC:antigencomplexes. Moreover, these modified RTLs, when associated with a cognatepeptide Ag, bind T-cells in an epitope-specific manner, and regulate Tcell activity (e.g., proliferation) in an Ag-specific manner, both invitro and in vivo. As a result, the disclosed MHC molecules are usefulin a wide range of both in vivo and in vitro applications.

Various formulations of modified, monodisperse RTLs are provided by theinvention. In exemplary embodiments, a modified RTL comprises a twodomain MHC class II component comprising α1 and β1 domains of amammalian MHC class II molecule. In more detailed embodiments, thesemodified RTLs are further characterized by having the amino terminus ofthe α1 domain covalently linked to the carboxy terminus of the β1domain. In other detailed embodiments, the MHC component of the RTL doesnot include α2 or β2 domains found in an intact MHC class II molecule.Typically, the MHC component of the RTL is associated, by covalent ornon-covalent interaction, with an antigenic determinant, such as a Tcell epitope of an autoantigenic protein. For example, a peptide antigenmay be covalently linked to the amino terminus of the β1 domain of aclass II MHC component. The two domain molecules may also comprise adetectable marker, such as a fluorescent label, or a toxic moiety (e.g.,ricin A).

The invention also provides nucleic acid molecules that encode theinventive, non-aggregating RTLs, as well as expression vectors that maybe used to express these molecules in mammalian cells. In particularembodiments, the nucleic acid molecules include sequences that encodethe MHC component as well as an antigenic peptide. For example, one suchnucleic acid molecule may be represented by the formula Pr—P—B-A,wherein Pr is a promoter sequence operably linked to P (a sequenceencoding the peptide antigen). B is the class II β1 domain, and A is theclass II α1 domain. In these nucleic acid molecules, P, B and A comprisea single open reading frame, such that the peptide and the two MHCdomains are expressed as a single polypeptide chain.

The modified RTLs of the invention may be used in vivo to detect andquantify T-cells, and/or to regulate T cell function. Specifically, suchmolecules loaded with a selected antigen may he used to detect, monitorand quantify the population of antigen-specific T cells, providingutility, inter alia, in a number of clinical settings, such asmonitoring the number of tumor antigen-specific T cells in blood removedfrom a cancer patient, or the number of self-antigen specific T cells inblood removed from a patient suffering from an autoimmune disease. Inthese contexts, the disclosed molecules are powerful tools formonitoring the progress of a particular therapy.

In addition to monitoring and quantifying antigen-specific T cells, themodified RTL molecules of the invention have utility for purifying Tcells for adoptive immunotherapy. For example, modified RTLs loaded witha tumor antigen may be used to purify tumor-antigen specific T cellsfrom a cancer patient. These cells may then be expanded in vitro beforebeing returned to the patient as part of an adoptive immunotherapeuticcancer treatment.

The modified RTL molecules of the invention can be used to alter theactivity, phenotype, differentiation status, and/or pathogenic potentialof T cells in an Ag-specific manner. Within alternate aspects of theinvention, these novel reagents can be used to induce a variety of Tcell transduction processes, to modulate T cell effector functions(including cytokine and proliferation responses), to induce anergy, orotherwise alter the pathogenic potential of T cells in an Ag-specificmanner. In this regard, the modified RTLs of the invention displaypowerful and epitope-specific effects on T-cell activation resulting, asexemplified by their ability to stimulate secretion of anti-inflammatorycytokines (e.g., IL-10). When conjugated with a toxic moiety, themodified RTLs of the invention may also be used to kill T cells having aparticular Ag specificity. Accordingly, the disclosed RTL molecules areuseful in a wide range of both in vivo and in vitro applications.

Modified RTL molecules of the invention can be readily produced byrecombinant expression in prokaryotic or eukaryotic cells, and can bepurified in large quantities. Moreover, these molecules may easily beloaded with any desired peptide antigen, making production of arepertoire of MHC molecules with different T-cell specificities a simpletask. These and other aspects of the invention are described in moredetail herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows HLA-DR2, RTL302, and the solvent accessible surface of theRTL β-sheet platform. The left panel (A) shows a scale model of an MHCclass II molecule on the surface of an APC. The right panel (B) showsRTL302, a soluble single-chain molecule derived from theantigen-binding/T cell recognition domains. The lower right panel (C)shows the hydrophobic residues of the beta-sheet platform of RTL302.

FIG. 2 shows size exclusion chromatography of modified RTLs. Purifiedand refolded RTLs were analyzed by size exclusion chromatography (SEC).The upper panel (A) shows SEC of RTL302 (triangle), RTL302(5S) (circle)and RTL302(5D) (square). These RTLs do not contain covalently tetheredAg-peptides. The lower panel (B) shows SEC of RTLs derived from thewild-type HLA-DR2 containing covalently tethered Ag-peptide MBP-85-99(RTL303, triangle) or MOG-35-55 (RTL312, circle).

FIG. 3 shows circular dichroism (CD) spectra of modified DR2-derivedRTLs. The upper panel (A) shows CD spectra of “empty” RTL302 (triangle),RTL302(5S) (circle) and RTL302(5D) (square). The middle panel (B) showsCD spectra of RTLs containing covalently tethered Ag MBP-85-99 peptide.RTL303, (triangle), RTL320 (square), and RTL340 (diamond). The lowerpanel (C) shows thermal denaturation curves for RTL303, RTL320 andRTL340 which reveal a high degree of cooperativity and stability.

FIG. 4 shows direct measurement of peptide binding to HLA-DR2-derivedRTLs. Binding of biotinylated-MOG to RTL302 (open circles), RTL302(5S)(open diamonds), and RTL302(5D) (open squares). The left panel (A) showssaturation as a function of biotinylated-MOG concentration (insert showsScatchard analysis of peptide binding). The right panel (B) showsbinding of biotinylated-MOG peptide (0.15 μM) to RTLs as a function oftime to compare the initial rate of binding.

FIG. 5 shows that monomeric, monodisperse RTL342 was as effective asRTL312 at treating EAE in DR*1501 transgenic animals. Mean clinicalscores of HLA-DR2 (DRB1*1501/DRA*0101) transgenic mice treated with 33μg of RTL312 (v), RTL342 (Δ), or vehicle alone (Tris, pH 8.5) (). Allmice were immunized s.c. with 200 μg MOG-35-55 and 400 μg CFA inconjunction with 100 ng Ptx i.v. on Day 0 and 266 ng Ptx 2 dayspost-immunization. On Day 14 all mice were distributed into 6 groupsaccording to similarity in disease and gender. Mice were i.v. injecteddaily with RTL312, RTL342, or vehicle. (n=4 per group, except forvehicle group where n=3; arrows indicate treatment).

FIG. 6 shows the interaction surface between the α1β1 peptide binding/Tcell recognition domain and the α2β2-Ig-fold domains of HLA-DR2. Theinteraction surface between the α1β1 peptide binding/T cell recognitiondomain and the α2β2-Ig-fold domains was modeled and refined using thehigh resolution human class II DR2 structure 1 BX2 (Smith et al., J.Exp. Med. 188:1511-20, 1998). The transmembrane domains are shownschematically as 0.5 nm cylinders. The amino and carboxyl termini of MHCclass II are labeled N, C, respectively. Cysteines are rendered asball-and-stick, as are the five residues V102, I104, A106, F108, L11O(1BX2 numbering). The interaction surface (4 angstrom interface) betweenthe Ig-fold domains and the peptide binding/T cell recognition domain iscolored by lipophilic potential (LP). Water molecules within thisinterface in the 1BX2 crystal structure are shown as spheres.

DEFINITIONS

The term “MHC” refers to the major histocompatibility complex.

The term “RTL” or “RTLs” refers to human recombinant T cell receptorligands.

“Ag” refers to antigen.

“APC” refers to antigen-presenting cell.

“β-ME” refers to β-mercaptoethanol.

“CD” refers to circular dichroism.

“CFA” refers to complete Freunds adjuvant.

“DLS” refers to dynamic light scattering.

“EAE” refers to experimental autoimmune encephalomyelitis.

“ELISA” refers to enzyme linked immunosorbant assay.

“HLA” refers to human leukocyte antigen.

“hu-” refers to human.

“MBP” refers to myelin basic protein.

“MHC” refers to major histocompatibility complex.

“MOG” refers to myelin oligodendrocyte glycoprotein, (murine sequence).

“MS” refers to multiple sclerosis.

“NFDM” refers to non fat dry milk.

“PBMC” refers to peripheral blood mononuclear cells.

“PBS” refers to phosphate-buffered saline.

“PCR” refers to polymerase chain reaction.

“Ptx” refers to pertussis toxin.

“RPMI” refers to growth media for cells developed at Rosweli ParkMemorial Institute.

“RT” refers to room temperature.

“RTL” refers to recombinant T cell receptor ligand (e.g., RTLs of G. G.Burrows, U.S. Pat. No. 6,270,772).

“S.C.” refers to subcutaneous.

“SEC” refers to size exclusion chromatography.

“STR-HRP” refers to streptavidin-horseradish peroxidase conjugate.

“TCR” refers to T cell receptor.

“Tg” refers to transgenic.

“Sequence identity” refers to the similarity between amino acidsequences. Sequence identity is frequently measured in terms ofpercentage identity (or similarity or homology); the higher thepercentage, the more similar the two sequences are. Variants of theinventive MHC domain polypeptides will possess a high degree of sequenceidentity when aligned using standard methods.

“MHC domain polypeptide” refers to a discrete MHC molecular domain, forexample an α1 or β1 domain of an MHC class II molecule.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS OF THE INVENTION

Modified RTLs of the invention comprise a major histocompatibilitycomplex (MHC) component that incorporates one or more redesigned surfacestructural features which have been recombinantly introduced into anotherwise native MHC polypeptide sequence. Typically, modified RTLs ofthe invention are rationally designed and constructed to introduce oneor more amino acid changes at a solvent-exposed target site locatedwithin, or defining, a self-binding interface found in the native MHCpolypeptide.

The self-binding interface that is altered in the modified RTL typicallycomprises one or more amino acid residue(s) that mediate(s)self-aggregation of a native MHC polypeptide, or of an “unmodified” RTLincorporating the native MHC polypeptide. Although the self-bindinginterface is correlated with the primary structure of the native MHCpolypeptide, this interface may only appear as an aggregation-promotingsurface feature when the native polypeptide is isolated from the intactMHC complex and incorporated in the context of an “unmodified” RTL.

Thus, in certain embodiments, the self-binding interface may onlyfunction as a solvent-exposed residue or motif of an unmodified RTLafter the native polypeptide is isolated from one or more structuralelement(s) found in an intact MHC protein. In the case of exemplary MHCclass II RTLs described herein (e.g., comprising linked β1 and α1domains), the native β1α1 structure only exhibits certainsolvent-exposed, self-binding residues or motifs after removal ofIg-fold like, β2 and α2 domains found in the intact MHC II complex.These same residues or motifs that mediate aggregation of unmodifiedβ1α1 RTLs, are presumptively “buried” in a solvent-inaccessibleconformation or otherwise “masked” (i.e., prevented from mediatingself-association) in the native or progenitor MHC II complex (likelythrough association with the Ig-fold like, β2 and α2 domains).

Certain modified RTLs of the invention include a multi-domain structurecomprising selected MHC class I or MHC class II domains, or portions ofmultiple MHC domains that are necessary to form a minimal Agrecognition/TCR interface (i.e., which is capable of mediating Agbinding and TCR recognition). In certain embodiments, the modified RTLcomprises a “minimal TCR interface”, meaning a minimal subset of MHCclass I or MHC class II domain residues necessary and sufficient tomediate functional peptide binding and TCR-recognition. TCR recognitionrequires that the modified RTL be capable of interacting with the TCR inan Ag-specific manner to elicit one or more TCR-mediated T cellresponses, as described herein.

In the case of modified RTLs derived from human class II MHC molecules,the RTLs will most often comprise α1 and β1 MHC polypeptide domains ofan MHC class II protein, or portions thereof sufficient to provide aminimal TCR interface. These domains or subportions thereof may becovalently linked to form a single chain (Sc) MHC class II polypeptide.Such RTL polypeptides are hereinafter referred to as “α1β1” sc MHC classII polypeptides. Equivalent sc MHC constructs can be modeled from humanMHC class I proteins, for example to form RTLs comprising α1 and α2domains (or portions thereof sufficient to provide a minimal TCRinterface) of a class I MHC protein, wherein the RTL is optionally“empty” or associated with an Ag comprising a CD8+ T cell epitope.

RTL constructs comprising sc MHC components have been shown to be widelyuseful for such applications as preventing and treating Ag-inducedautoimmune disease responses in mammalian model subjects predictive ofautoimmune disease therapeutic activity in humans (Burrows et al., J.Immunol. 161:5987, 1998; Burrows et al., J. Immunol. 164:6366, 2000). Inother aspects, these types of RTLs have been demonstrated to inhibit Tcell activation and induce anti-inflammatory cytokine (e.g., IL-10)secretion in human DR2-restricted T cell clones specific for MBP-85-95or BCR-ABL b3a2 peptide (CABL) (Burrows et al., J. Immunol. 167:4386,2001; Chang et al., J. Biol. Chem. 276:24170, 2001).

Additional RTL constructs have been designed and tested by inventors inthe instant application, which include a MOG-35-55/DR2 construct (VG312)shown to potently inhibit autoimmune responses and lead to immunologicaltolerance to the encephalitogenic MOG-35-55 peptide and reverse clinicaland histological signs of EAE (Vandenbark et al., J. Immunol.171:127-33, 2003). Numerous additional RTL constructs that are usefulfor modulating T cell immune responses and can be employed within theinvention are available for use within the methods and compositions ofthe invention (see, e.g., U.S. Pat. No, 5,270,772, issued Aug. 7, 2001;U.S. Provisional Patent Application No. 60/064,552, filed Sep. 16, 1997;U.S. Provisional Patent Application No. 60/064,555, filed Sep. 16, 1997;U.S. Provisional Patent Application No. 60/200,942, filed May 1, 2000;U.S. Provisional Patent Application entitled MONOMERIC RECOMBINANT MHCMOLECULES USEFUL FOR MANIPULATION OF ANTIGEN-SPECIFIC T-CELLS, filed byBurrows et al. on Sep. 5, 2003 and identified by Attorney Docket No.49321-98; U.S. patent application Ser. No. 09/153,586; filed May 1,2001; U.S. patent application Ser. No. 09/847,172; filed May 1, 2001;and U.S. patent application Ser. No. 09/858,580; filed May 15, 2001,each incorporated herein by reference).

To evaluate the biological function and mechanisms of action of modifiedRTLs of the invention, antigen-specific T cells hearing cognate TCRshave been used as target T cells for various assays (see, e.g., Burrowset al., J. Immunol. 167:4386, 2001). More recently, inventors in thecurrent application have provided novel T cell hybridomas that areuniquely adapted for use in screens and assays to identify andcharacterize RTL structure and function (see, e.g., U.S. ProvisionalPatent Application No. 60/586,433, filed Jul. 7; and Chou et al., J.Neurosci. Res. 77: 670-680, 2004). To practice these aspects of theinvention, T cell hybrids are constructed and selected that display anAg-specific, TCR-mediated proliferative response following contact ofthe hybrid with a cognate Ag and APCs. This proliferative response of Thybrids can in turn be detectably inhibited or stimulated by contactingthe T cell hybrid with a modified RTL of interest, which yields amodified, Ag-specific, TCR-mediated proliferation response of thehybrid. The modified proliferation response of the hybrid cellaccurately and reproducibly indicates a presence, quantity, and/oractivity level of the modified RTL in contact with the T cell hybrid.

The MHC component of the RTL may be provided as an “empty” RTL, or beassociated by non-covalent binding or covalent linkage to a selectedpeptide Ag. Typically, the peptide Ag comprises one or more antigenicdeterminant(s) of an autoantigenic protein, for example one or more CD4+T cell immunodominant epitope(s) associated with a selected autoimmunedisease (e.g., an immunodominant epitope of myelin basic protein (MBP)or myelin oligodendrocyte protein (MOG) implicated in MS).

Within certain embodiments of the invention, an isolated, modifiedrecombinant RTL which has a reduced potential for aggregation insolution comprises an “MHC component” in the form of a single chain (sc)polypeptide that includes multiple, covalently-linked MHC domainelements. These domain elements are typically selected from a) α1 and β1domains of an MHC class II polypeptide, or portions thereof comprisingan Ag-binding pocket/T cell receptor (TCR) interface; or b) α1 and α2domains of an MHC class I polypeptide, or portions thereof comprising anAg-binding pocket/TCR interface. The MHC component of the RTL ismodified by one or more amino acid substitution(s), addition(s),deletion(s), or rearrangement(s) at a target site corresponding to a“self-binding interface” identified in a native MHC polypeptidecomponent of an unmodified RTL. The modified RTL modified exhibits amarkedly reduced propensity for aggregation in solution compared toaggregation exhibited by an unmodified, control RTL having the samefundamental MHC component structure, but incorporating the native MHCpolypeptide defining the self-binding interface.

As used herein, “native MHC polypeptide” refers to intact,naturally-occurring MHC polypeptides, as well as to engineered orsynthetic fragments, domains, conjugates, or other derivatives of MHCpolypeptides that have an identical or highly conserved amino acidsequence compared to an aligned sequence in the naturally-occurring MHCpolypeptide (e.g., marked by 85%, 90%, 95% or greater amino acididentity over an aligned stretch of corresponding residues. The “nativeMHC polypeptide” having the self-associating interface will often be anMHC polypeptide domain incorporated within an unmodified RTL, and theself-associating interface may only be present in such a context, asopposed to when the native MHC polypeptide is present in a fully intact,native MHC protein (e.g., in a heterodimeric MHC class II proteincomplex).

Thus, in the case of MHC class II RTLs, removal of the β2 and α2 domainsto create a smaller, more useful (e.g., β1α1 ) domain structure for theRTL (comprising a minimal TCR interface) results in “unmasking” (i.e.,rendering solvent-exposed) certain self-binding residues or motifs thatcomprise target sites for RTL modification according to the invention.These unmasked residues or motifs can be readily altered, for example bysite-directed mutagenesis, to reduce or eliminate aggregation and renderthe RTL as a more highly monodisperse reagent in aqueous solution.

To evaluate the extent of monodispersal of these modified RTLs, anunmodified or “control” RTL may be employed which has the same basicpolypeptide construction as the modified RTL, but features the nativeMHC polypeptide sequence (having one or more amino acid residues ormotifs comprising the self-binding interface and defining asolvent-exposed target site for the modification when the nativepolypeptide is incorporated in the RTL).

The modified RTLs of the invention yield an increased percentage ofmonodisperse molecules in solution compared to a corresponding,unmodified RTL (i.e., comprising the native MHC polypeptide and bearingthe unmodified, self-binding interface). In certain embodiments, thepercentage of unmodified RTL present as a monodisperse species inaqueous solution may he as low as 1%, more typically 5-10% or less oftotal RTL protein, with the balance of the unmodified RTL being found inthe form of higher-order aggregates. In contrast, modified RTLs of thepresent invention will yield at least 10%-20% monodisperse species insolution. In other embodiments, the percentage of monomeric species insolution will range from 25%-40%, often 50%-75%, up to 85%, 90%, 95% orgreater of the total RTL present, with a commensurate reduction in thepercentage of aggregate RTL species compared to quantities observed forthe corresponding, unmodified RTLs under comparable conditions.

The self-binding interface that is altered in the MHC polypeptide toform the modified RTL may comprise single or multiple amino acidresidues, or a defined region, domain, or motif of the MHC polypeptide,which is characterized by an ability to mediate self-binding orself-association of the MHC polypeptide and/or RTL. As used herein,“self-binding” and “self-association” refers to any intermolecularbinding or association that promotes aggregation of the MHC polypeptideor RTL in a physiologically-compatible solution, such as water, saline,or serum.

As noted above, MHC class II molecules comprise non-covalentlyassociated, α- and β-polypeptide chains. The α-chain comprises twodistinct domains termed α1 and α2. The β-chain also comprises twodomains, β1 and β2. The peptide binding pocket of MHC class II moleculesis formed by interaction of the α1 and β1 domains. Peptides fromprocessed antigen bind to MHC molecules in the membrane distal pocketformed by the β1 and α1 domains (Brown et al., 1993; Stern et al.,1994). Structural analysis of human MHC class II/peptide complexes(Brown et al., Nature 364:33-39, 1993; Stern et al., Nature 368:215,1994) demonstrate that side chains of bound peptide interact with“pockets” comprised of polymorphic residues within the class II bindinggroove. The bound peptides have class II allele-specific motifs,characterized by strong preferences for specific amino acids atpositions that anchor the peptide to the binding pocket and a widetolerance for a variety of different amino acids at other positions(Stern et al., Nature 368:215, 1994; Rammensee et al., Immunogenetics41: 178, 1995). Based on these properties, natural populations of MHCclass II molecules are highly heterogeneous. A given allele of class IImolecules on the surface of a cell has the ability to bind and presentover 2000 different peptides. In addition, bound peptides dissociatefrom class II molecules with very slow rate constants. Thus, it has beendifficult to generate or obtain homogeneous populations of class IImolecules bound to specific antigenic peptides.

The α2 and β2 domains of class II molecules comprise distinct,trans-membrane Ig-fold like domains that anchor the α- and β-chains intothe membrane of the APC. In addition, the α2 domain is reported tocontribute to ordered oligomerization during T cell activation (Konig etal., J. Exp. Med. 182:778-787, 1995), while the β2 domain is reported tocontain a CD4 binding site that co-ligates CD4 when the MHC-antigencomplex interacts with the TCR αβ heterodimer (Fleury et al., Cell66:1037-1049, 1991; Cammarota et al., Nature 356:799-801, 1992; Kanig etal., Nature 356:796-798, 1992; Huang et al., J. Immunol. 158:216-225,1997).

RTLs modeled after MHC class II molecules for use within the inventiontypically comprise small (e.g., approximately 200 amino acid residues)molecules comprising all or portions of the α1 and β1 domains of humanand non-human MHC class II molecules, which are typically geneticallylinked into a single polypeptide chain (with and without covalentlycoupled antigenic peptide). Exemplary MHC class II-derived “β1α1”molecules retain the biochemical properties required for peptide bindingand TCR engagement (including TCR binding and/or partial or complete TCRactivation). This provides for ready production of large amounts of theengineered RTL for structural characterization and immunotherapeuticapplications. The MHC component of MHC class II RTLs comprise a minimal,Ag-binding/T cell recognition interface, which may comprise all orportions of the MHC class II α1 and β1 domains of a selected MHC classII molecule. These RTLs are designed using the structural backbone ofMHC class II molecules as a template. Structural characterization ofRTLs using circular dichroism indicates that these molecules retain anantiparallel β-sheet platform and antiparallel α-helices observed in thecorresponding, native (i.e., wild-type sequence) MHC class IIheterodimer. These RTLs also exhibit a cooperative two-state thermalfolding-unfolding transition. When the RTL is covalently linked with Agpeptide they often show increased stability to thermal unfoldingrelative to empty RTL molecules.

In exemplary embodiments of the invention, RTL design is rationallybased on crystallographic coordinates of human HLA-DR, HLA-DQ, and/orHLA-DP proteins, or of a non-human (e.g., murine or rat) MHC class IIprotein. In this context, exemplary RTLs have been designed based oncrystallographic data for HLA DR1 (PDB accession code 1AQD), whichdesign parameters have been further clarified, for example, by sequencealignment with other MHC class H molecules from rat, human and mousespecies. The program Sybyl (Tripos Associates, St Louis, Mo.) is anexemplary design tool that can be used to generate graphic images using,for example, an O2 workstation (Silicon Graphics, Mountain View, Calif.)and coordinates obtained for HLA-DR, HLA-DQ, and/or HLA-DP molecules.Extensive crystallographic characterizations are provided for these andother MHC class II proteins deposited in the Brookhaven Protein DataBank (Brookhaven National Laboratories, Upton, N.Y.).

Detailed description of HLA-DR crystal structures for use in designingand constructing modified RTLs of the invention is provided, forexample, in Ghosh et al., Nature 378:457, 1995; Stern et al., Nature368:215, 1994; Murthy et al., Structure 5:1385, 1997; Bolin et al., J.Med. Chem. 43:2135, 2000; Li et al., J. Mol. Biol. 304:177, 2000;Hennecke et al., Embo J. 19:5611, 2000; Li et al., Immunity 14:93, 2001;Lang et al., Nat. Immunol. 3:940, 2002; Sundberg et al., J. Mol. Biol.319:449, 2002; Zavala-Ruiz et al., J. Biol. Chem. 278:44904, 2003;Sundberg et al., Structure 11:1151, 2003. Detailed description of HLA-DQcrystal structures is provided, for example, in Sundberg et al., Nat.Struct. Biol. 6:123, 1999; Li et al., Nat. Immunol. 2:501, 2001; andSiebold et al., Proc. Nat. Acad. Sci. USA 101:1999, 2004. Detaileddescription of a murine MHC I-A^(U) molecule is provided, for example,in He et al., Immunity 17:83, 2002. Detailed description of a murine MHCclass II I-Ad molecule is provided, for example, in Scott et al.,Immunity 8:319, 1998. Detailed description of a murine MHC class II I-Akmolecule is provided, for example, in Reinherz et al., Science 286:1913,1999, and Miley et al., J. Immunol. 166:3345, 2001. Detailed descriptionof a murine MHC allele I-A(G7) is provided, for example, in corner etal., Science 288:501, 2000. Detailed description of a murine MHC classII H2-M molecule is provided, for example, in Fremont et al., Immunity9:385, 1998. Detailed description of a murine MHC class II H2-Ieβmolecule is provided, for example, in Krosgaard et al., Mol. Cell12:1367, 2003; Detailed description of a murine class II Mhc I-Abmolecule is provided, for example, in Zhu et al., J. Mol. Biol.326:1157, 2003. HLA-DP Lawrance et al., Nucleic Acids Res. 1985 Oct. 25;13(20): 7515-7528

Structure-based homology modeling is based on refined crystallographiccoordinates of one or more MHC class I or class II molecule(s), forexample, a human DR molecule and a murine I-E^(k) molecule. In oneexemplary study by Burrows and colleagues (Protein Engineering12:771-778, 1999), the primary sequences of rat, human and mouse MHCclass II were aligned, from which it was determined that 76 of 256α-chain amino acids were identical (30%), and 93 of the 265 β-chainamino acids were identical (35%). Of particular interest, the primarysequence location of disulfide-bonding cysteines was conserved in allthree specks, and the backbone traces of the solved structures showedstrong homology when superimposed, implying an evolutionarily conservedstructural motif, with side-chain substitutions designed to allowdifferential antigenic-peptide binding in the peptide-binding groove.

Further analysis of MHC class I and class II molecules for constructingmodified RTLs of the invention focuses on the “exposed” (i.e., solventaccessible) surface of the β-sheet platform/anti-parallel α-helix thatcomprise the domain(s) involved in peptide binding and T cellrecognition. In the case of MHC class II molecules, the α1 and β1domains exhibit an extensive hydrogen-bonding network and a tightlypacked and “buried” (i.e., solvent inaccessible) hydrophobic core. Thistertiary structure is similar to molecular features that conferstructural integrity and thermodynamic stability to the α-helix/β-sheetscaffold characteristic of scorpion toxins, which therefore present yetadditional structural indicia for guiding rational design of modifiedRTLs herein (see, e.g., Zhao et al., J. Mol. Biol. 227:239, 1992;Housset, J. Biol. 238:88-91, 1994; Zinn-Justin et al., Biochemistry35:8535-8543, 1996).

From these and other comparative data sources, crystals of native MHCclass II molecules have been found to contain a number of watermolecules between a membrane proximal surface of the β-sheet platform inand a membrane distal surfaces of the α2 and β2 Ig-fold domains.Calculations regarding the surface area of interaction between domainscan be quantified by creating a molecular surface, for example for theβ1α1 and α2β2 Ig-fold domains of an MHC molecule, using an algorithmsuch as that described by Connolly (Biopolymers 25:1229-1247, 1986) andusing crystallographic coordinates (e.g., as provided for various MHCclass II molecules in the Brookhaven Protein Data Base.

For an exemplary, human DR1 MHC class II molecule (PDB accession numbers1SEB, 1AQD), surface areas of the β1α1 and α2β2-Ig-fold domains werecalculated independently, defined by accessibility to a probe of radius0.14 nm, about the size of a water molecule (Burrows et al., ProteinEngineering 12:771-778, 1999). The surface area of the MHC class IIαβ-heterodimer was 156 nm², while that of the β1α1 construct was 81 nm²and the α1β2-Ig-fold domains was 90 nm². Approximately 15 nm² (18.5%) ofthe β1α1 surface was found to be buried by the interface with theIg-fold domains in the MHC class II αβ-heterodimer. Side-chaininteractions between the β1α1-peptide binding and Ig-fold domains (α2and β2) were analyzed and shown to be dominated by polar interactionswith hydrophobic interactions potentially serving as a “lubricant” in ahighly flexible “ball and socket” type inter face.

These and related modeling studies suggest that the antigen bindingdomain of MHC class II molecules remain stable in the absence of the α2and β2 Ig-fold domains, and this production has been born out forproduction of numerous, exemplary RTLs comprising an MHC class II “α1β1”architecture. Related findings were described by Burrows et al. (J.Immunol. 161:5987-5996, 1998) for an “empty” β1α1 RTL, and four α1β1 RTLconstructs with covalently coupled rat and guinea pig antigenicpeptides: β1 1-Rt-MBP-72-89, β1 1-Gp-MBP-72-89, β1 1-Gp-MBP-55-69 and β11Rt-CM-2. For each of these constructs, the presence of native disulfidebonds between cysteines (β15 and β79) was demonstrated by gel shiftassay with or without the reducing agent β-mercaptoethanol (β-ME). Inthe absence of β-ME, disulfide bonds are retained and the RTL proteinstypically move through acrylamide gels faster due to their more compactstructure. These data, along with immunological findings using MHC classII-specific monoclonal antibodies to label conserved epitopes on theRTLs generally affirm the conformational integrity of RTL moleculescompared to their native MHC II counterparts (Burrows et al., 1998,supra; Chang et al., J. Biol. Chem. 276:24170-14176, 2001; Vandenbark etal., J. Immunol. 171:127-133, 2003). Similarly, circular dichroism (CD)studies of MHC class II-derived RTLs reveal that β1α1 molecules havehighly ordered secondary structures. Typically, RTLs of this generalconstruction shared the β-sheet platform/anti-parallel α-helix secondarystructure common to all class II antigen binding domains. In thiscontext, β1α1 molecules have been found to contain, for example,approximately 30% α-helix, 15% β-strand, 26% β-turn and 29% random coilstructures. RTLs covalently bound to Ag peptide (e.g., MBP-72-89, andCM-2) show similar, although not identical, secondary structuralfeatures. Thermal denaturation studies reveal a high degree ofcooperativity and stability of RTL molecules, and the biologicalintegrity of these molecules has been demonstrated in numerous contexts,including by the ability of selected RTLs to detect and inhibit ratencephalitogenic T cells and treat experimental autoimmuneencephalomyelitis.

According to these and related findings provided herein (or described inthe cited references which are collectively incorporated herein for alldisclosure purposes), RTL constructs of the invention, with or withoutan associated antigenic peptide, retain structural and conformationalintegrity consistent with that of refolded native MHC molecules. Thisgeneral finding is exemplified by results for soluble single-chain RTLmolecules derived from the antigen-binding/TCR interface comprised ofall or portions of the MHC class II β1 and α1 domains. In more detailedembodiments, these exemplary MHC class II RTLs lack the α2 domain and β2domain of the corresponding, native MHC class II protein, and alsotypically exclude the transmembrane and infra-cytoplasmic sequencesfound in the native MHC II protein. The reduced size and complexity ofthese RTL constructs, exemplified by the “β1α1” MHC II RTL constructs,provide for ready and predictable expression and purification of the RTLmolecules from bacterial inclusion bodies in high yield (e.g., up to15-30 mg/1 cell culture or greater yield).

In native MHC class II molecules, the Ag peptide binding/T cellrecognition domain is formed by well-defined portions of the α1 and β1domains of the α and β polypeptides which fold together to form atertiary structure, most simply described as a β-sheet platform uponwhich two anti-parallel helical segments interact to feint anantigen-binding groove. A similar structure is formed by a single exonencoding the α1 and α2 domains of MHC class I molecules, with theexception that the peptide-binding groove of MHC class II is open-ended,allowing the engineering of single-exon constructs that encode thepeptide binding/T cell recognition domain and an antigenic peptideligand.

As exemplified herein for MHC class H proteins, modeling studieshighlighted important features regarding the interface between the β1α1and α2β2-Ig-fold domains that have proven critical for designingmodified, monodisperse RTLs of the invention. The α1 and β1 domains showan extensive hydrogen-bonding network and a tightly packed and “buried”(i.e., solvent inaccessible) hydrophobic core. The β1α1 portion of MHCclass II proteins may have the ability to move as a single entityindependent from the α2β2-Ig-fold ‘platform’. Besides evidence of a highdegree of mobility in the side-chains that make up the linker regionsbetween these two domains, crystals of MHC class II I-Ek contained anumber of water molecules within this interface (Jardetzky et al.,Nature 368: 711-715, 1994; Fremont et al., Science 272:1001-1004, 1996;Murthy et al., Structure 5:1385, 1997). The interface between the β1α1and α2β2-Ig-fold domains appears to be dominated by polar interactions,with hydrophobic residues likely serving as a ‘lubricant’ in a highlyflexible ‘ball and socket’ type interface. Flexibility at this interfacemay be required for freedom of movement within the α1 and β1 domains forbinding/exchange of peptide antigen. Alternatively or in combination,this interaction surface may play a role in communicating informationabout the MHC class II peptide molecular interaction with TCRs back tothe APC.

Following these rational design guidelines and parameters, the instantinventors have successfully engineered modified, monodispersederivatives of single-chain human RTLs comprising peptide binding/TCRrecognition portions of human MHC class II molecules (e.g., asexemplified by a HLA-DR2b (DRA*0101/DRB1*1501). Unmodified RTLsconstructed from the α1 and β1 domains of this exemplary MHC class IImolecule retained biological activity, but formed undesired, higherorder aggregates in solution.

To resolve the problem of aggregation in this exemplary, unmodified RTL,site-directed mutagenesis was directed towards replacement ofhydrophobic residues with polar (e.g., serine) or charged (e.g.,aspartic acid) residues to modify the β-sheet platform of theDR2-derived RTLs. According to this rational design procedure, novel RTLvariants were obtained that were determined to be predominantlymonomeric in solution. Size exclusion chromatography and dynamic lightscattering demonstrated that the novel modified RTLs were monomeric insolution, and structural characterization using circular dichroismdemonstrated a highly ordered secondary structure of the RTLs.

Peptide binding to these “empty”, modified RTLs was quantified usingbiotinylated peptides, and functional studies showed that the modifiedRTLs containing covalently tethered peptides were able to inhibitantigen-specific T cell proliferation in vitro, as well as suppressexperimental autoimmune encephalomyelitis in vivo. These studiesdemonstrated that RTLs encoding the Ag-binding/TCR recognition domain ofMHC class II molecules are innately very robust structures. Despitemodification of the RTLs as described herein, comprising site-directedmutations that modified the β-sheet platform of the RTL, these moleculesretained potent biological activity separate from the Ig-fold domains ofthe progenitor class II structure, and exhibited a novel and surprisingreduction in aggregation in aqueous solutions. Modified RTLs havingthese and other redesigned surface features and monodispersalcharacteristics retained the ability to bind Ag-peptides, inhibit T cellproliferation in an Ag-specific manner,and treat, inter alia, autoimmunedisease in vivo.

Additional modifications apart from the foregoing surface featuremodifications can be introduced into modified RTLs of the invention,including particularly minor modifications in amino acid sequence(s) ofthe MHC component of the RTL that are likely to yield little or nochange in activity of the derivative or “variant” RTL molecule.Preferred variants of non-aggregating MHC domain polypeptides comprisinga modified RTLs are typically characterized by possession of at least50% sequence identity counted over the full length alignment with theamino acid sequence of a particular non-aggregating MHC domainpolypeptide using the NCBI Blast 2.0, gapped blastp set to defaultparameters. Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 90% or at least 95% sequence identity. Whenless than the entire sequence is being compared for sequence identity,variants will typically possess at least 75% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are known in the art as described above. Variants ofmodified RTLs comprising non-aggregating MHC domain polypeptides alsoretain the biological activity of the non-variant, modified RTL. For thepurposes of this invention, that activity may be conveniently assessedby incorporating the variation in the appropriate MHC component of amodified RTL (e.g., a β1α1 MHC component) and determining the ability ofthe resulting RTL/Ag complex to inhibit Ag-specific T-cell proliferationin vitro, as described herein.

Rationally Designed Mutations Converted Complexes of Human Recombinant TCell Receptor Ligands Into Monomers That Retain Biological Activity

Applicant's herein demonstrate and disclose that the potent biologicalactivity of particular RTLs (Burrows et al., J. Immunol. 167:4386-95;2001; Wang et al., The Journal of Immunology, 2003; Vandenbark et al.,Journal of Immunology, 2003) was retained when produced in a monomericform, with the ability to inhibit T cell proliferation in vitro.Extremely important from a clinical perspective, the monomeric form isable to reverse clinical signs of EAE and induce long-term T celltolerance against the encephalitogenic, DR2-restricted, MOG-35-55peptide in Tg mice that uniquely express this multiplesclerosis-associated HLA-DR2 allele.

Applicant's earlier studies had demonstrated that immunization of Tg-DR2mice with MOG-35-55 peptide induced strong T cell responses,perivascular spinal cord lesions with demyelination, and severe chronicsigns of EAE, as well as anti-MOG antibodies that were apparently notinvolved in either disease or tolerance induction (Vandenbark et al.,Journal of Immunology, 2003).

As disclosed herein, treatment of the Tg-DR2 mice after onset ofclinical EAE with an 8-day course of daily i.v. injections of 33 μgRTL342 reversed disease progression to baseline levels and maintainedreduced clinical activity even after cessation of further injections.Treatment with control RTL303 containing covalently tethered MBP-87-99did not inhibit EAE or affect T cell responses to MOG-35-55 peptide,demonstrating antigen specificity.

Significantly, the applicant's teachings are the first to document thatmonomeric RTLs have such potent clinical activity, and that themolecules are suitable for evaluation for use in human clinical trialsfor treatment of multiple sclerosis.

The peptide binding/TCR recognition domain of MHC class II from whichRTLs are derived contain a complex mixture of alpha-helix and beta-sheetsecondary structure, as well as a highly conserved post-translationalmodification, a disulfide bond between cysteines at position 16 and 80(RTL302 numbering). These molecules are small enough to systematicallydissect with currently available technology, yet complex enough thatsuccessful engineering of other MHC molecules and derivatives,comprehensive of HLA-DR, HLA-DQ, and HLA-DP molecules and derivatives,will require application of the novel protein engineering findings andconcepts disclosed herein.

MHC class II molecules have (at least) three clearly defined biochemical“functions” that can be used to evaluate and quantify the retention of aspecific three-dimensional fold derived from the primary sequence:Ag-peptide binding, TCR binding and CD4 binding. Without being bound bytheory, applicant's hypothesized that these functions have been encodedand superimposed onto the primary sequence of MHC class II, and thatsome of these functions can be separated experimentally for evaluationusing protein engineering (Burrows et al., Protein Engineering12:771-78, 1999; Chang et al., J. Biol. Chem. 276:24170-76, 2001).

For purposes of the present invention, it is desirable to retain two keybiochemical functions: the ability to specifically bind Ag-peptides; andthe ability to bind the αβ heterodimer chains of the TCR. Retention ofthese key features allows discernment of the minimal interactioninterface with the T cell that still initiates a throughput informationsignal (Wang et al., The Journal of Immunology, 2003), allowingengineering a molecular system for controlling CD4⁺ T cells in anAg-specific manner.

While HLA-DR2-derived RTLs with the wild-type sequence retained thesetwo key biological activities (Vandenbark et al., Journal of Immunology,2003), they tended to form higher-order structures (Burrows et al., J.Immunol 167:4386-95, 2001) that could not be completely eliminated bymanipulating solvent conditions. For example, an optimal yield ofmonodisperse monomeric RTL302 of almost 20% was obtained by decreasingthe concentration of purified RTL302 protein to 0.1 mg/ml for the finalfolding step, and changing buffers from phosphate-buffered saline toTris. However, concentrating purified RTL302 monomer above 0.2 mg/mlcaused the molecules to repartition back into a mixture of monomer andaggregate, an equilibrium that was concentration dependent. According tothe present invention, aggregation of HLA-DR2 derived RTLs is specificto certain portions of the DR2-derived RTL sequence.

A 2.6 angstrom resolution crystal structure of HLA-DR2 with boundAg-peptide MBP-85-99 (PDB accession 1BX2; (Smith et al., J. Exp. Med.188:1511-20, 1998), provided sufficient data to permit analysis hereinof the membrane-proximal surface of the β-sheet platform and themembrane distal surfaces of the α2 and β2 Ig-fold domains, specificallyidentifying features that contribute to higher-order structures oraggregation when the subject MHC II domains are incorporated in anunmodified RTL. Specifically, according to the present invention, theβ-sheet platform buried in the progenitor HLA-DR2 molecule defines thebottom of the RTLs, and contains a number of hydrophobic residues thatare typically found buried within a protein structure rather than beingsolvent exposed.

The propensity of different amino acid residues to be present in β-sheetstructures has been intensively investigated (Minor et al., Nature367:660-63, 1994; Pokkuluri et al., Protein Science 11:1687-94, 2002;Street et al., Proc. Natl. Acad. Sci. USA 96:9074-76; 1999; Chou et al.,Biochemistry 13:211-22, 1973; Smith et al., Biochemistry 33:5510-17,1994; Finkelstein, Protein Engineering 8:207-09, 1995), as part of anoverall goal of understanding the rules that dictate secondary structurestability and formation. The body of work available has defined themarkedly high preference in β-sheets for the β-branched amino acidsisoleucine, valine, and threonine, as well as aromatic amino acidresidues phenylalanine and tyrosine.

According to the present invention, desired surface modification of anRTL comprising an MHC class II component to yield much less aggregationprone form can be achieved, for example, by replacement of one or morehydrophobic residues identified in the β-sheet platform of the MHCcomponent with non-hydrophobic residues, for example polar or chargedresidues. Modified RTL constructs exemplifying this aspect of theinvention were constructed by replacing one or more target, hydrophobicresidues identified in the β-sheet platform of an HLA-DR2 component ofRTL 302 with one or more exemplary polar (e.g., serine) residue(s) and,alternatively exemplary charged (e.g., aspartate) residue(s). FIG. 1depicts the targeted β-sheet platform residues for modification.Initially, a central core portion of the β-sheet platform was targetedfor modification, comprising V102, I104, A106, F108, and L110 (shownfrom top to bottom in FIG. 1C). These residues were changed bysite-directed mutagenesis, individually, or in various multiple-residuecombinations to either a serine, or aspartate residue(s).

Individual changes of these hydrophobic residues to a different, polaror charged residue, generally yielded detectable reductions inaggregation of the modified RTL. The more hydrophobic residues that werechanged, either to a polar or charged residue, the greater the expectedreduction in aggregation potential. When all five of the indicated, coreβ-sheet platform target residues were changed, a substantial conversionof the RTLs to a more monodisperse form was observed. When all five coretarget residues were changed to a serine residue, approximately 15% ofthe modified RTLs were rendered monomeric in solution. When all fivecore target residues (comprising an exemplary, self-binding “motif” ofthe DR molecule) were changed to apartate residues, substantially all ofthe modified RTLs were observed in a monodisperse form in solution (seebelow). As illustrated in FIG. 1C, additional hydrophobic targetresidues are available for modification to alter self-bindingcharacteristics of the β-sheet platform portion of DR2 moleculesincorporated in RTLs. In reference to FIG. 1C, the left arm of thediagrammed β-sheet platform includes a separate “motif” of three notedhydrophobic residues (top to bottom), L141, V138, and A133 that willserve as useful targets for individual or collective modification (e.g.,by site directed amino acid deletion or substitution) to remove thehydrophobic residue(s) or alter the target residue(s) to anon-hydrophobic (e.g., polar, or charged) residue. Also in reference toFIG. 1C, several target hydrophobic residues are marked to the right ofthe core β-sheet motif, including L9, F19, L28, F32, V45, and V51, whichmay be regarded as one or more additional, self-binding orself-associating target “motifs” for RTL modification. Any one orcombination of these residues may be targeted for modification to removethe hydrophobic residue(s) or alter the target residue(s) to anon-hydrophobic residue, with the expectation of yielding furtherbenefits with regard to diminishing aggregation potential of themodified RTL.

RTL modification typically involves amino acid substitution or deletionat target sites for mutagenesis comprising a self-binding interface(comprised of one or more amino acid residues, or a self-binding motifformed of several target residues). Within exemplary embodimentsdirected toward production of modified RTLs that comprise MHC class IIRTL components, targeted residues for modification typically comprisehydrophobic residues or motifs, for example valine, leucine, isoleucine,alanine, phenylalanine, tyrosine, and tryptophan. These and other targetresidues may be advantageously deleted, or alternatively substituted forany non-hydrophobic amino acid. Suitable substituent amino acids forgenerating desired RTL modifications can include amino acids havingaliphatic-hydroxyl side chains, such as serine and threonine; aminoacids having amide-containing side chains, such as asparagine andglutamine; amino acids having aromatic side chains, such asphenylalanine, tyrosine, and tryptophan; and amino acids having basicside chains, such as lysine, arginine, and histidine.

The findings presented herein validate the rational design approach ofthe current invention for modifying a diverse array of RTLs comprisingvarious MHC components, including MHC class I and MHC class IIcomponents, to diminish the propensity of the modified RTL foraggregation in solution. These concepts are clearly applicable to alltypes of MHC class II molecules, which exhibit a high degree ofconservation of higher order structure, including particularly in theβ-sheet platform portion of the molecule that is solvent-exposed throughproduction of RTLs (e.g., comprising β1α1 MHC class II sc polypeptide)The beta strand of the HLA-DR2 component successfully modified inexemplary RTLs herein defines an extended, central strand within theprogenitor HLA-DR2 molecule (FIG. 6). The 2.6 angstrom resolutioncrystal structure of HLA-DR2 with bound Ag-peptide MBP-85-99 (PDBaccession 1BX2; (Smith et al., J. Exp. Med. 188:1511-20, 1998)),permitted analysis herein of the membrane-proximal surface of theβ-sheet platform and the membrane distal surfaces of the α2 and β2Ig-fold domains, specifically identifying features that contributed tohigher-order structures or aggregation. Specifically, according to thepresent invention, the beta-sheet platform buried in the progenitorHLA-DR2 molecule defines the bottom of the RTLs, and contains a numberof hydrophobic residues that are typically found buried within a proteinstructure rather than being solvent exposed.

All human HLA-DR molecules will follow closely the modification rulesand expectations for rational design disclosed herein. Likewise, humanHLA-DQ and HLA-DP class II molecules, and various murine, rat and othermammalian class II MHC molecules, are described and characterizedsufficiently (see references cited above, which are incorporated herein)to apply the structure-function analytical rules of the inventionrelating to identification and modification of RTL self-bindinginterface residues and motifs to these related subjects within themethods and compositions of the invention.

In the case of exemplary, modified, DR2-derived RTLs, the greatestsuccess in terms of rendering monodisperse RTL derivatives was obtainedby substitution of all five β-sheet platform core hydrophobic residueswith an exemplary charged residue, aspartate. Aspartate is significantlyunder-represented in β-sheet regions of proteins, and the introductionherein of either five serine, or five aspartate residues on the externalface of an interior strand of the β-sheet platform of RTLs had only asubtle effect on the secondary structure as quantified by circulardichroism (FIG. 3). This moderate effect was interpreted as anapproximately 10% increase of anti-parallel β-strand structure upondeconvolution of the spectra (Table III).

A likely explanation for why these exemplary modified RTLs maintain thesame basic fold and biological features as their counterpart, unmodifiedRTLs bearing the native MHC components and self-binding residues/motifs,comes from analysis of the β-sheet in the context of its functional roleas an “open” platform in the overall tertiary structure of theAg-binding/TCR recognition domain of the progenitor HLA-DR2 molecule,rather than a closed surface like an Ig-fold. The propensity ofdifferent amino acid residues to be present in β-sheet structures hasbeen intensively investigated (Minor et al., Nature 367:660-63, 1994;Pokkuluri et al., Protein Science 11:1687-94, 2002; Street et al., Proc.Natl. Acad. Sci. USA 96:9074-76; 1999; Chou et al., Biochemistry13:211-22, 1973; Smith et al., Biochemistry 33:5510-17, 1994;Finkelstein, Protein Engineering 8:207-09, 1995), as part of an overallgoal of understanding the rules that dictate secondary structurestability and formation. The body of work available has defined themarkedly high preference in β-sheets for the β-branched amino acidsisoleucine, valine, and threonine, as well as aromatic amino acidresidues phenylalanine and tyrosine.

To illustrate the broad applicability of the rational design principlesand methods of the invention for constructing modified, monodisperseRTLs from a diverse array of MHC components, the following alignment ispresented documenting homologous self-binding motifs identified withindifferent, exemplary RTLs constructed with homologous MHC componentsfrom HLA-DR, HLA-DP, and HLA-DQ MHC class II molecules.

Primary amino acid sequence alignment of human RTLs.RTL302 was derived from DR2 (DRB1*15011/DRA*0101) (31).RTL600 was derived from DP2 (DPA1*0103/DPB1*0201).RTL800 was derived from DQ2 (DQA1*05/DQB1*02) (**Italics indicate non-native amino acid residues. Gaps in the sequences for optimalalignment (-) and the beta 1//alpha 1 junction (↓) are also shown.The conserved cysteines that form a disulfide bond are shaded yellow.

The foregoing alignment maps a homologous self-binding motif identifiedand modified herein for an exemplary HLA-DR2 component in RTL302 inalignment with homologous target residues comprising self-binding motifson additional RTLs constructed by the present inventors, includingRTL600, comprising a DP2 MHC component, and RTL800, comprising a DQ2 MHCcomponent. Consistent with the disclosure and teachings herein, numerousexemplary target residues of RTL600 and RTL800 are readily discerned(e.g., as matched with target DR2 residues marked by underscoring in thealignment) for modification of these additional DQ and DP MHC class IIspecies to produce modified RTLs according to the invention.

With respect to designing modified RTLs containing an MHC class IIcomponent, FIG. 6 shows the interaction surface between the oil α1β1peptide binding/T cell recognition domain and the α2β2-Ig-fold domainsof HLA-DR2. The interaction surface between the α1β1 peptide binding/Tcell recognition domain and the α2β2-Ig-fold domains was modeled andrefined using the high resolution human class II DR2 structure 1BX2(Smith et al., J. Exp. Med. 188:1511-20, 1998). The transmembranedomains are shown schematically as 0.5 nm cylinders. The amino andcarboxyl termini of MHC class II are labeled N, C, respectively.Cysteines are rendered as ball-and-stick, as are the five residues V102,I104, A106, F108, L110 (1BX2 numbering). The interaction surface (4angstrom interface) between the Ig-fold domains and the peptidebinding/T cell recognition domain is colored by lipophilic potential(LP). Water molecules within this interface in the 1BX2 crystalstructure are shown as red spheres.

Within certain embodiments of the invention, substitution of one or moretarget hydrophobic residue(s) with one or more charged residue(s)(exemplified here by aspartate) provides the additional objective ofconstraining the modified RTL molecule to stay extended or “stretched”out along this interior β-strand by charge-charge repulsion, rather thanallowing the structure to collapse onto itself in the absence of theIg-fold domains that are present in the progenitor HLA-DR2 molecule.This conclusion is consistent with the data herein, and is additionallysupported by reports that β-sheet propensity for an amino acid residuearises from local steric interactions of the amino acid side chain withits local backbone (Minor et al., Nature 367:660-63, 1994).

These instant results demonstrate that RTLs encoding the Ag-binding/TCRrecognition domain of MHC class II molecules are innately very robuststructures, capable of retaining activity separate from the Ig-folddomains of the progenitor class II structure, and even after fairlyaggressive modification to make the molecules monomeric andmonodisperse. Applying the methods and tools of the invention, increasedsolubility and prevention of aggregation of modified RTLs is readilyaccomplished by modification of a self-binding interface, motif, orresidue(s), exemplified by an exposed surface of a native MHC class IIstructure that was originally buried in the progenitor proteinstructure. By staying within thermodynamic limitations that constrainthe protein's final folded structure and by not interfering with theprocess by which the protein domain achieves this final fold, a keyobstacle to recombinant design of monodisperse RTLs has beenovercome—which is the requirement to leave intact within the primarysequence the “code” that drives folding toward a final unique structurethat retains the ability to bind peptides and bind the TCR in anAg-peptide-specific manner, retaining potent biological activity.

The following sections provide detailed guidance on the design,expression and uses of recombinant MHC molecules of the invention.Unless otherwise stated, standard molecular biology, biochemistry andimmunology methods are used. Such standard methods are described inSambrook et al. (1989), Ausubel et al (1987), Innis et al. (1990) andHarlow and Lane (1988). The following U.S. patents and additionalpatents and publications cited elsewhere herein relate to design,construction and formulation of MHC molecules and their uses, and areincorporated herein by reference to provide additional background andtechnical information relevant to the present invention: U.S. Pat Nos.5,130,297; 5,194,425; 5,260,422; 5,284,935; 5,468,481; 5,595,881;5,635,363; 5,734,023.

Design of Recombinant MHC Class II β1α1 Molecules

The amino acid sequences of mammalian MHC class II α and β chainproteins, as well as nucleic acids encoding these proteins, are wellknown in the art and available from numerous sources, includingreferences cited and incorporated herein, and GenBank. Within exemplaryembodiments of the invention, modified RTLs may comprise astructurally-reduced or minimized MHC component that exclude all or partof a native MHC protein, for example all or part of an MHC class II β2domain, or at least a CD4-binding portion of the β2 domain. Alsotypically excluded from the modified RTL is an α2 domain of the MHCclass II protein. RTLs of this construction can be “empty” (i.e., freeof peptide Ag), or non-covalently bound or covalently linked to peptideAg. In more detailed aspects of the invention, the α1 and β1 MHC classII domains comprising the modified RTL may be linked together to foam asingle chain (sc) polypeptide (Burrows et al., Protein Engineering12:771-78; 1999; Chang et al., J. Biol. Chem. 276:24170-76, 2001).

Among the modified RTLs provided within the invention are exemplaryconstructs comprising recombinant MHC class II molecules, which inexemplary embodiments include the β1 domain of the MHC class II β chaincovalently linked to the α1 domain of the MHC class II α chain. The β1and α1 domains are well defined in mammalian MHC class II proteins.Typically, the α1 domain is regarded as comprising about residues 1-90of the mature a chain. The native peptide linker region between the α1and α2 domains of the MHC class II protein spans from about amino acid76 to about amino acid 93 of the a chain, depending on the particular αchain under consideration. Thus, an α1 domain may include about aminoacid residues 1-90 of the a chain, but one of skill in the art willrecognize that the C-terminal cut-off of this domain is not necessarilyprecisely defined, and, for example, might occur at any point betweenamino acid residues 70-100 of the a chain. The composition of the α1domain may also vary outside of these parameters depending on themammalian species and the particular a chain in question. One of skillin the art will appreciate that the precise numerical parameters of theamino acid sequence are much less important than the maintenance ofdomain function.

Similarly, the β1 domain is typically regarded as comprising aboutresidues 1-90 of the mature βb chain. The linker region between the β1and β2 domains of the MHC class II protein spans from about amino acid85 to about amino acid 100 of the β chain, depending on the particularchain under consideration. Thus, the β1 protein may include about aminoacid residues 1-100, but one of skill in the art will again recognizethat the C-terminal cut-off of this domain is not necessarily preciselydefined, and, for example, might occur at any point between amino acidresidues 75-105 of the β chain. The composition of the β1 domain mayalso vary outside of these parameters depending on the mammalian speciesand the particular β chain in question. Again, one of skill in the artwill appreciate that the precise numerical parameters of the amino acidsequence are much less important than the maintenance of domainfunction.

Nucleic acid molecules encoding these domains may be produced bystandard means, such as amplification by the polymerase chain reaction(PCR). Standard approaches for designing primers for amplifying openreading frames encoding these domain may be employed. Libraries suitablefor the amplification of these domains include, for example, cDNAlibraries prepared from the mammalian species in question; suchlibraries are available commercially, or may be prepared by standardmethods. Thus, for example, constructs encoding the β1 and α1polypeptides may be produced by PCR using four primers: primers β1 andβ2 corresponding to the 5′ and 3′ ends of the β1 coding region, andprimers A1 and A2 corresponding to the 5′ and 3′ ends of the α1 codingregion. Following PCR amplification of the α1 and β1 domain codingregions, these amplified nucleic acid molecules may each be cloned intostandard cloning vectors, or the molecules may be ligated together andthen cloned into a suitable vector. To facilitate convenient cloning ofthe two coding regions, restriction endonuclease recognition sites maybe designed into the PCR primers. For example, primers B2 and A1 mayeach include a suitable site such that the amplified fragments may bereadily ligated together following amplification and digestion with theselected restriction enzyme. In addition, primers B1 and A2 may eachinclude restriction sites to facilitate cloning into the polylinker siteof the selected vector. Ligation of the two domain coding regions isperformed such that the coding regions are operably linked, i.e., tomaintain the open reading frame. Where the amplified coding regions areseparately cloned, the fragments may be subsequently released from thecloning vector and gel purified” preparatory to ligation.

In particular embodiments, a peptide linker is provided between the β1and α1 domains. Typically, this linker is between 2 and 25 amino acidsin length, and serves to provide flexibility between the domains suchthat each domain is free to fold into its native conformation. Thelinker sequence may conveniently he provided by designing the PCRprimers to encode the linker sequence. Thus, in the example describedabove, the linker sequence may be encoded by one of the B2 or A1primers, or a combination of each of these primers.

Nucleic acid expression vectors including expression cassettes will beparticularly useful for research purposes. Such vectors will typicallyinclude sequences encoding the dual domain MHC polypeptide (β1 α1) witha unique restriction site provided towards the 5′ terminus of the MHCcoding region, such that a sequence encoding an antigenic polypeptidemay be conveniently attached. Such vectors will also typically include apromoter operably linked to the 5′ terminus of the MHC coding region toprovide for high level expression of the sequences.

In particular embodiments, β1 α1 molecules may also be expressed andpurified without an attached peptide, in which case they may be referredto as “empty.” The empty MHC molecules may then be loaded with theselected peptide as described below.

Sequence Variants. One of skill in the art will appreciate that variantsof the disclosed inventive molecules and domains may be made andutilized in the same manner as described. Thus, reference herein to adomain of an MHC polypeptide or molecule (e.g., an MHC class II β1domain) includes both preferred forms of the referenced molecule, aswell as molecules that are based on the amino acid sequence thereof, butwhich include one or more amino acid sequence variations. Such variantpolypeptides may also be defined in the degree of amino acid sequenceidentity that they share with the disclosed preferred molecule.Typically, MHC domain variants will share at least 80% sequence identitywith the sequence of the preferred MHC domains disclosed herein. Morehighly conserved variants will share at least 90% or at least 95%sequence identity with the preferred MHC domains disclosed herein.Variants of MHC domain polypeptides also retain the biological activityof the preferred MHC domains disclosed herein. For the purposes of thisinvention, that activity is conveniently assessed by incorporating thevariant domain in the appropriate β1α1 polypeptide and determining theability of the resulting polypeptide to inhibit antigen specific T-cellproliferation in vitro, as described in detail herein below.

Variant MHC domain polypeptides include proteins that differ in aminoacid sequence from the preferred MHC domains disclosed herein, but whichretain the specified biological and non-aggregating activity. Suchproteins may be produced by manipulating the nucleotide sequence of themolecule encoding the domain, for example by site-directed mutagenesisor the polymerase chain reaction. The simplest modifications involve thesubstitution of one or more amino acids for amino acids having similarbiochemical properties. These so-called conservative substitutions arelikely to have minimal impact on the activity of the resultant protein.Table I shows amino acids which may be substituted for an original aminoacid in a protein and which are regarded as conservative substitutions,which are well known in the art.

More substantial changes in biological function or other features may beobtained by selecting substitutions that are less conservative, i.e.,selecting residues that differ more significantly in their effect onmaintaining (a) the structure of the polypeptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain. The substitutions which in general areexpected to produce the greatest changes in protein properties will bethose in which (a) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histadyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine. The effects ofthese amino acid substitutions or deletions or additions may be assessedthrough the use of the described T-cell proliferation assay.

At the nucleic acid level, one of skill in the art will appreciate thatthe naturally occurring nucleic acid sequences that encode class I andII MHC domains may be employed in the expression vectors, but that theinvention is not limited to such sequences. Any sequence that encodes afunctional MHC domain may be employed, and the nucleic acid sequence maybe adapted to conform with the codon usage bias of the organism in whichthe sequence is to be expressed.

Modified RTLs of the invention exhibit a reduced capacity forself-aggregation compared to a corresponding, umnodified RTL (i.e., anRTL comprising only native MHC amino acid sequences). Therefore, therational design of RTL surface modifications described herein yield anincreased percentage of the RTL molecules present as monomers insolution compared to a monodisperse fraction of unmodified RTLs insolution.

Despite the surface structural changes introduced into the modifiedRTLs, these novel RTL constructs retain the ability to specifically bindAg-peptides and to functionally interact with a TCR on a target T cell.Exemplary functions of the modified RTLs include an ability to modulateT cell activity in an Ag-specific manner to reduce a pathogenicpotential or alter a pathogenic phenotype of the T cell (e.g., toinhibit T cell proliferation, reduce inflammatory cytokine production bythe T cell, or increase anti-inflammatory cytokine production by the Tcell). As such, the modified RTLs have utility to treat autoimmunediseases and other conditions mediated by antigen-specific T cells invivo.

Within other aspects of the invention, modified RTLs are provided thatlack trans-membrane Ig fold domains found in an intact MHC molecule,wherein the modified RTLs are non-aggregating or exhibit reducedaggregation compared to unmodified RTLs. The modified RTLs possess Tcell regulatory activity as described herein, despite lacking certainstructural features present in the corresponding, intact MHC molecule.MEC components of modified RTLs of the invention refold in a manner thatis structurally analogous to native whole MHC molecules, and the bindpeptide antigens to fin m stable MHC-antigen complexes.

Desired T cell responses that can be elicited, individually, in variouscombinations, or collectively, by the modified RTLs of the inventiontypically comprise one or more phenotypic change(s) that correlate(s)with an altered or “reprogrammed” state of a T cell associated withreduced pathogenic activity or potential (e.g., a reduced ability tomediate or potentiate autoimmune tissue destruction or other symptom(s)diagnostic of a selected autoimmune disease).

Modified RTL molecules of the invention are useful, for example, todetect, quantify and purify Ag-specific T-cells. In other embodiments,the modified RTLs are useful in compositions and methods to modulate Tcell phenotype, activity, differentiation status, migration behavior,tissue localization, and/or cell fate. Within these aspects of theinvention, compositions and methods are provided for modulating one ormore T cell activities selected from T cell activation, proliferation,and/or expression of one or more cytokine(s), growth factor(s),chemokines, cell surface receptors (e.g., TCRs), and/or cellularadhesion molecules (CAMs). Properly designed, evaluated andadministered, modified RTLs of the invention serve as potent T cellregulatory agents, in vitro or in vivo, to quantitatively modulate orqualitatively “switch” T cell phenotype-particularly with respect topathogenic potential of the target T cell.

By contacting a target T cell with a modified RTL of the invention(bearing cognate antigen bound or linked to the TCR interface in theabsence of costimulatory, factors (e.g., APCs and other regulatorysignals as conferred by native, MHC II α2 and β2 regulatory sequences),the compositions and methods of the invention can function to“reprogram” a target T cell to alter the differentiation status or fateof the T cell, for example to an induced, nonpathogenic state orphenotype characterized by reduced pathogenic potential. For example,modified RTLs of the invention can be employed to reprogram a T cellfrom an original, pathogenic pathway of differentiation to one thatyields a “T suppressor” phenotype. In additional embodiments RTLs of theinvention can be employed to reprogram a T cell by eliciting a “switch”in one or more cytokines, or in a “cytokine expression profile”, forexample a switch from a Th1 to a Th2 cytokine expression profile, whichin turn provides methods to reprogram T-cells to treat or manageautoimmune diseases and other disorders mediated by the T-cells.Additional description of these and related aspects of the invention isprovided by Huan et al., J. Immunol. 172:4556-4566, 2004 (incorporatedherein by reference).

Further uses for modified RTL constructs of the invention include, forexample, evaluating T cell activity and function, or TCR function andbinding specificity, in diagnostic and analytic contexts (see, e.g.,Wang et al., J. Immunol.) In more specific embodiments, RTLs of theinvention can be used for detection, quantification and/or purificationof T-cells that recognize particular antigens to yield importantdiagnostic and therapeutic information and materials. By way of example,early detection of T-cells specific for a particular autoantigen using,for example a labeled RTL, will facilitate early selection ofappropriate treatment regimes. The ability to purify antigen-specificT-cells will also be of great value in adoptive immunotherapy. Adoptiveimmunotherapy involves the removal of T-cells, e.g., from a cancerpatient, expansion of the T-cells in vitro and then reintroduction ofthe cells to the patient (see U.S. Pat. No. 4,690,915; Rosenberg et al.New Engl. J. Med. 319: 1676-1680 (1988)). Isolation and expansion ofcancer specific T-cells with inflammatory properties will increase thespecificity and effectiveness of immunological intervention.

In more detailed aspects of the invention, modified RTLs comprising aMHC class I or class II component is used in vivo to target and alter apathogenic potential or activity of Ag-specific T cells. By way ofexample, a β1α1 molecule loaded with a T cell Ag (e.g., an antigenicepitope, domain, region, peptide, or other portion, derivative orconjugate of a T cell antigenic protein) and administered to patientssuffering from multiple sclerosis or other autoimmune disease may beused to modulate T cell activity (e.g., modulate T cell proliferation,modulate T cell expression of one or more cytokine(s), chemokine(s),growth factor(s), and/or adhesion or other cell migratory factor(s), orinduce anergy and/or phenotypic change(s) in T cell fate,differentiation status, location, bystander signaling or suppressionactivity, and/or pathogenic potential) in Ag-specific T cells—therebyalleviating or preventing associated disease symptoms. Alternatively,such molecules may be conjugated with a toxic moiety to directly killdisease-causing T cells.

The following examples set forth to provide those of ordinary skill inthe art with a more detailed disclosure and description of the subjectinvention, and are not intended to limit the scope of what is regardedas the invention. Efforts have been made to ensure accuracy with respectto the numbers used (e.g., amounts, temperature, concentrations, etc.)but certain experimental errors and deviations should be allowed for.

EXAMPLE 1 Methods

Homology modeling, Much of the logic for dissecting the molecules hasbeen previously described (Burrows et al., Protein Engineering12:771-78, 1999; Chang et al., J. Biol. Chem. 276:24170-76, 2001).Sequence alignment of MHC class II molecules from human, rat and mousespecies provided a starting point for our studies and graphic imageswere generated with the program Sybyl 6.9 (Tripos Associates, St. Louis,Mo.) on an O2 workstation (IRIX 6.5, Silicon Graphics, Mountain View,Calif.) using coordinates deposited in the Brookhaven Protein Data Bank(Brookhaven National Laboratories, Upton, N.Y.). Structure-basedhomology modeling was based on the refined crystallographic coordinatesof human HLA-DR2 (Smith et al., J. Exp. Med. 188:1511-20, 1998; Li etal., J. Mol. Biol. 304:177-88, 2000), as well as DR1 (Brown et al.,Nature 364:33-39, 1993; Murthy et al., Structure 5:1385-96, 1997),murine I-E^(k) molecules (Fremont et al., Science 272:1001-04, 1996),and scorpion toxins (Zhao et al., J. Mol. Biol. 227:239-52, 1992;Housset et al., J. Mol. Biol. 238:88-91, 1994; Zinn-Justin et al.,Biochemistry 35:8535-43, 1996). Amino acid residues in human HLA-DR2(PDB accession code 1BX2) were used. This structure was determined bysingle wavelength diffraction and molecular replacement (AmoRe XRay/NMRstructure refinement package, C.N.R.S., France) using HLA-DR1 as astarting structure (PDB accession code 1DLH) (Stern et al., Nature368:215, 1994). The following residues were either missing or hadmissing atoms in the final structure: chain A; K2, M36, K38, K39, E46,N78, R100, E101; chain B: E22, E35, E52, E59, K65, E69, P108, R189,(1BX2 numbering) (Smith et al., J. Exp. Med. 188:1511-20, 1998). Forthese residues the correct side chains were inserted and the peptidebackbone was modeled as a rigid body during structural refinement usinglocal energy minimization.

RTL structural modification. De novo synthesis of human HLA-DR2 derivedRTLs has been previously described (Chang et al., J. Biol. Chem.276:24170-76, 2001). Site-directed mutagenesis was used to replacehydrophobic residues on the solvent accessible surface of the β-sheetplatform of the RTLs with polar (serine) or charged (aspartic acid)residues. The modification was performed by using the QuickChange™site-directed mutagenesis method as described by Stratagene (La Jolla,Calif.). In brief, PCR reaction with Pfu DNA polymerase (Stratagene, LaJolla, Calif.) was performed by using RTL302 or RTL303 as template andtwo synthetic oligonucleotide primers containing the desiredmutation(s). For example, a pair of mutation primers for RTL320 were 1)forward primer: 5′-GGC GAG TCA TCA AAG AAG AAC ATA GCA TCA GCC AGA GCGAGA GTT ATA GTA ATC CTG ACC AAT C-3′; 2) backward primer: 5′-GAT TGG TCAGGA TTA CTA TAA CTC TCG CTC TGG CTG ATG CTA TGT TCT TCT TTG ATG ACTC-3′; and a pair of mutation primers for RTL340 were 1) forward primer:5′-GGC GAG TCA TCA AAG AAG AAC ATG ACA TCG ACC AGG ACG AGG ACT ATG ACAATC CTG ACC AAT C-3′; 2) backward primer. 5′-GAT TGG TCA GGA TTG TCA TAGTCC TCG TCC TGG TCG ATG TCA TGT TCT TCT TTG ATG ACT C-3′. Theoligonucleotide primers, each complementary to the opposite strand oftemplate, were extended during 19 temperature cycles by means of Pfu DNApolymerase at an annealing temperature of 55° C. Upon incorporation ofthe oligonucleotide primers, a mutated plasmid containing staggerednicks is generated. Following temperature cycling, the PCR product wastreated with DpnI endonuclease to digest the parental DNA template andto select for mutants containing the DNA sequence of interest. Thenicked plasmid DNA incorporating the desired mutation(s) was thentransformed into E. Coli BK21(DE3) as an expression host (Novagen,Madison, Wis.). Colonies were screened and cells containing plasmid withthe desired mutation(s) were used for plasmid purification using QIAprepSpin Miniprep kit (QIAGEN, Valencia, Calif.). The purified plasmid DNAwas then digested with NcoI and XhoI to confirm the efficiency ofmutation. Finally, the desired plasmids were sequenced with the (T7)5′-TAA TAC GAC TCA CTA TAG GG-3 and (T7 terminal) 5′-GCT AGT TAT TGC TCAGCG G-3′ primers to confirm mutations of interest.

Expression and refolding of soluble RTL molecules. Expression,purification and refolding of human HLA-DR2 derived RTLs was previouslydescribed (Chang et al., J. Biol. Chem 276:24170-76, 2001). A number ofmodifications have been made in the protocol to streamline productionwhile maintaining or slightly increasing the yield of protein. Bacteriawere grown in one liter cultures to mid-logarithmic phase(OD₆₀₀=0.6-0.7) in Luria-Bertani (LB) broth containing carbenicillin (50μg/ml) at 37° C. Recombinant protein production was induced by additionof 0.5 mM isopropyl β-D-thiogalactoside (IPTG). After incubation for 4hours, the cells were harvested by centrifugation and stored at 4° C.(short-term) or −80° C. (long-term) before processing. All subsequentmanipulations of protein purification were at 4° C. The cell pelletswere resuspended in lysis buffer (50 mM Tris-Cl, 0.1 M NaCl, 5 mM EDT A,pH 7.4). Lysozyme (10 mg/ml solution in lysis buffer; 1 mg per gram ofcell pellet) was added, and the solution was incubated at roomtemperature for 30 minutes, swirling gently every 10 minutes. The cellsuspension was then sonicated for 6×5 seconds with the cell suspensioncooled in a salt ice water bath. The cell suspension was centrifuged(20,000 g for 10 minutes at 4° C., Beckman J2-21, JA-14 rotor), thesupernatant fraction was poured off, the cell pellet resuspended andwashed two times in 100 ml lysis buffer containing 1% Triton X-100 andthen one wash in lysis buffer without Triton X-100, and then resuspendedin 100 ml Buffer A (20 mM ethanolamine, 6 M urea, pH 10), and stirredgently at 4° C. overnight. After centrifugation (40,000 g for 45 minutesat 4° C., Beckman J2-21, JA-20 rotor), the supernatant containing thesolubilized recombinant protein of interest was filtered (0.22μStericup, Millipore) and stored at 4° C. until purification. Therecombinant proteins of interest were purified and concentrated by FPLCion-exchange chromatography using Source 30Q anion-exchange media(Pharmacia Biotech, Piscataway, N.J.) in an XK26/20 column (PharmaciaBiotech), using a step gradient with buffer A and buffer B (20 mMethanolarnine/HCl, 6M urea, 2M NaCl, pH 10.0). Fractions containing therecombinant protein of interest were pooled and concentrated for sizeexclusion chromatography (SEC buffer, 20 mM ethanolamine, 6M urea, 0.2 MNaCl, pH 10.0; column, Superdex 75, HRI6/60). Fractions containingprotein of interest were pooled and diluted with SEC buffer to OD₂₈₀ of0.1. Proteins were dialyzed against 20 mM Tris-Cl at pH 8.5, whichremoved the urea and allowed refolding of the recombinant protein.Following dialysis, the proteins were concentrated by centrifugalultrafiltration with Centricon10 membranes (Amicon, Beverly, Mass.). Forpurification to homogeneity, a finish step was included using sizeexclusion chromatography (Superdex 75, HRI6/60). The final yield ofpurified protein varied between 15 to 30 mg/L of bacterial culture.

SDS-gel shift assay. Aliquots of purified protein sample were denaturedby boiling for 5 min in Laemmli buffer with or without the reducingagent β-mercaptoethanol, and then analyzed by electrophoresis (12%SDS-PAGE). After electrophoresis, gels were stained with Coomassiebrilliant blue (Sigma, St. Louis, Mo.) and destained for observation ofmolecular weight shifting.

Dynamic light scattering. Dynamic light scattering (DLS) experimentswere conducted with a DynaPro™ instrument (Protein Solutions, Inc.,Charlottesville, Va.). The protein samples in 20 mM Tris-Cl buffer at pH8.5 were filtered through 100 nm Anodisc membrane filter (Whatman,Clifton, N.J.) at a concentration of 1.0 mg/ml and 20 μl of filteredsample were loaded into a quartz cuvette and analyzed at 488 Dm. Fiftyspectra were collected at 4° C. to get estimation of the diffusioncoefficient and relative polydispersity of proteins in aqueous solution.Data were then analyzed by Dynamics software version 525.44 (ProteinSolutions, Charlottesville, Va.) and buffer baselines were subtracted.Data were expressed as the mean of the calculated hydrodynamic radius.Molecular weights of RTLs were calculated assuming a globular hydratedshape for the molecules using Dynamics software version 5.25.44 (ProteinSolutions, Charlottesville, Va.).

Circular dichroism (CD) and thermal denaturation analysis. CD analysisand thermal denaturation studies were preformed as previously described(Chang et a., J. Biol. Chem 276:24170-76, 2001). In brief, recombinantproteins in 20 mM Tris-CI buffer pH 8.5 were analyzed using an AvivModel 215 CD spectrometer (Aviv Associates, Lakewood, N.J.). Spectrawere the average of 4-5 scans from 260 to 180 nm, recorded at a scanningrate of 5 nm/min with 4-second time constant. Data were collected at 0.5nm intervals. Spectra were averaged and smoothed using built-inalgorithms and buffer baselines were subtracted. Secondary structure wasestimated using a deconvolution software package (CDNN version 2.1, AvivAssociates, Lakewood, N.J.) based on the variable selection method(Compton et al., Analytical Biochemistry 155: 155-67, 1986). CD versustemperature (thermal denaturation curve) was recorded at a fixedwavelength of 208 nm. Temperature gradients from 60 to 95° C. weregenerated with a software controlled thermoelectric device to generaterising or falling temperature steps. Heating and cooling rates werebetween 10-12° C./h. The transition curves were normalized to 0 mdeg at60° C. and are plotted as the change in absorbance (mdeg) as a functionof temperature.

Enzyme linked immunosorbant assay (EL/SA). Biotinylated MOG-35-55peptide (Biot-MEVGWYRSPFSRVVHLYRNGK-OH), non-biotinylated MOG-35-55 andMBP-85-99 peptide (ENPVVHFFKNIVTPR-OH) were purchased from New EnglandPeptide, Inc., (Fitchburg, Mass.). The purity of the peptides wasverified by a reverse phase HPLC and mass identification was performedusing MALDI-TOF to verify mass was within 0.1% of molecular weightexpected. The peptides were lyophilized and stored at −80° C. until use.Direct binding assay experiments were carried out in order to determinethe ability of the RTLs to bind peptide and to determine theconcentration of the biotinylated peptide at which all specific bindingsites were saturated under the conditions used in our studies. ELISAplates (Maxisorp, Nunc, Rochester, N.Y.) were coated with 50 μl ofprotein at a concentration of 1 μg/ml in 20 mM Tris, pH 8.5 (50 ng ofprotein; i.e., 40 nM) overnight at 4° C., washed 4 times with washsolution (0.05% Tween 20, PBS, pH 7.4), and blocked with a Caseinsolution (BioFX, Owing Mills, Md.) for 1.5 h at room temperature. Plateswere then washed 4× and 50 μl of biotinylated peptide (serial dilutions)were added to the wells, RT, 1.5 h, and then washed 4×. 50 μl of astreptavidin-horseradish peroxidase conjugate (STR-HRP, 1:5000, DAKO,Glostrup, Denmark) in PBS was added to the wells and incubated at RT for1.5 h then washed 4× to remove unbound conjugate. 50 μl of HRP substrate(BioFX) was added for 45 min, RT. Reactions were stopped with StopSolution (BioFX) and bound peptide was determined indirectly by readingthe absorbance at 405 nm in an ELISA plate reader (Applied Biosystems,Molecular Devices, Sunnyvale, Calif.). A standard curve of STR-HRPconcentration vs OD₄₀₅ nm was used to determine the concentration ofbound peptide. To control nonspecific binding, wells were coated with 3%non fat dry milk (NFDM) in PBS and treated in the same way as theRTL-coated wells. In order to determine the time required to reachsteady-state binding of the peptides to the proteins, ELISA plates werecoated, washed and blocked as above and then biotinylated peptide inPBS/1 mM EDTA at pH 7.4 at 0.15 μM was added at different times (0 to 36h).

T cell clones and T cell proliferation assay. Antigen-specific T cellclones were selected from PBMC of an MS patient homozygous forHLA-DRB1*1501 as previously described (Burrows et al., J. Immunol167:4386-95, 2001). Selected antigen-specific T cell clones weresubcloned by limiting dilution method and subsequentially evaluated forantigen-specific proliferation (Burrows et al., J. Immunol. 167:4386-95,2001) The clone with the highest stimulation index (SI) was selected andcontinuously cultured in RPMI medium supplemented with 1% human serumand 5 ng/ml IL-2. The clonality of cells was determined by RT-PCR with aclone defined as a T cell population utilizing a single TCR Vβ gene(Burrows et al., J. Immunol 167:4386-95, 2001). T cell clones wereexpanded by stimulation with 1 μ/ml MOG-35-55 or MBP-85-99 peptide and2×10⁵ irradiated (2500 rad) autologous PBMCs per well in a 96-wellplate. The expanded T cells were maintained in 1% human serum RPMIcontaining 5 ng/ml IL-2. Fresh 1L-2 was added twice a week and T cellclones were restimulated with irradiated (2500 rad) autologous PBMCsevery three weeks. Antigen-specific T cell proliferation were performedperiodically to verify the quality of the cells. For these assays,antigen-specific T cell clones were washed twice with RPMI medium and5×10⁴ cells were re-seeded into each well in a 96-well plate andincubated in triplicate with 2×10⁵ freshly isolated and irradiated (2500rad) autologous PBMCs with 10 μg/ml of the desired peptide. Cells wereincubated for 72 hours with [³H]-thymidine added for the last 18 hours.Cells were harvested by a Harvester 96 (Tomtec, Hamden, Conn.) andradioactivity incorporated was measured on a 1205 BS liquidscintillation counter (Wallac, Turku, Finland), Stimulation index (SI)was calculated by dividing the mean cpm of peptide-added wells by themean cpm of the medium alone control wells. In RTL treatmentexperiments, 8 μM of the desired RTL was pre-incubated with the T cellclones for 72 hours, following by two washes with RPMI media before theT cell proliferation assay was performed.

Mice. HLA-DR2 Tg mice bearing chimeric MHC class II molecules weredeveloped as previously described (Woods et al., J. Exp. Med.180:173-81, 1994). The peptide-binding domain of MHC class II is encodedby human sequences while the membrane proximal portion including theCD4-binding domain is encoded by mouse sequences (DRα1*0101: I-Eα andDRβ1*1501: I-Eβ, previously described). The DRβ1*1501: I-Eβ constructwas made essentially as described in Woods et al. (Woods et al., J. Exp.Med. 180:173-81, 1994), with the following changes: The pACYCI84 vectorcontaining the DRB1*0401 exons 1 and 2, and the Eβ^(d) exons 3-6 waspartially digested with BamHI and treated with Klenow polymerase toremove a BamHI site in the vector. Subsequently, DRB1*1501 exon 2 wascloned into pACYC184 which had been predigested with BamHI and EcoRI toremove DRB1*0401 exon 2, Transgenic mice were generated bymicroinjecting the chimeric α- and β-chain constructs into fertilizedeggs from (DBA/2×C57BL/6)F₁ matings. Viable embryos were transferredinto pseudo pregnant females for development to term. Transgenicoffspring were backcrossed twice to the MHC class II knock out mouse,MHCII^(Δ/Δ) (Madsen et al., Pro Natl Acad Sci USA 96:10338-43, 1999).

Induction of active EAE and treatment with RTLs. Tg HLA-DR2 male andfemale mice between 8 and 12 weeks of age were immunized subcutaneouslyas described (Ito et al., J Immunol. 167, 2001) at four sites on theflanks with 0.2 ml of an emulsion comprised of 200 μg mouse MOG-35-55peptide in complete Freund's adjuvant (CFA) containing 400 μgMycobacterium tuberculosis H37RA (Difco, Detroit, Mich.). In addition,mice were given pertussis toxin (Ptx, List Biological Laboratories,Campbell, Calif.) on Day 0 and Day 2 post-immunization (25 ng and 67 ngper mouse, respectively). Mice were treated i.v. daily for 8 days,beginning 2-4 days after onset of clinical signs, with 100 μl of RTL312,RTL342, or vehicle (20 mM Tris, pH 8.5) containing 33 μg of the RTLproteins. Actively immunized mice were assessed daily for clinical signsof EAE according to the following scale: 0=normal; 1=limp tailor mildhind limb weakness; 2=limp tail and moderate hind limb weakness or mildataxia; 3=limp tail and moderately severe hind limb weakness; 4=limptail and severe hind limb weakness or mild forelimb weakness or moderateataxia; 5=limp tail and paraplegia with no more than moderate forelimbweakness; and 6=limp tail and paraplegia with severe forelimb weaknessor severe ataxia or moribund condition. The average daily score wasdetermined for each mouse by summing the daily clinical scores anddividing by the number of days the mouse exhibited clinical signs. Themean peak and average daily scores plus or minus SD were calculated forthe control and experimental groups.

EXAMPLE 2 Rationally Designed Mutations Converted Complexes of HumanRecombinant T Cell Receptor Ligands Into Monomers That RetainedBiological Activity

We have recently described protein engineering studies of recombinantTCR ligands (RTLs) derived from the alpha-1 and beta-1 domains ofHLA-DR2 (DRB1*1501/DRA*0101) (Chang et al., J. Biol. Chem. 276:24170-76,2001). These molecules formed well defined aggregates that were highlysoluble in aqueous buffers, with retention of biological activity(Burrows et al., J. Immunol. 167:4386-95, 2001; Buenafe, JBC, 2003;Vandenbark et al., Journal of Immunology, 2003). We analyzed themembrane proximal surface of the β-sheet platform that packed on themembrane distal surfaces of the α2 and β2 Ig-fold domains, specificallylooking for features that might contribute to higher-order structures oraggregation (FIG. 1).

FIG. 1 shows HLA-DR2, RTL302, and the solvent accessible surface of theRTL β-sheet platform. The left panel (A) shows a scale model of an MHCclass II molecule on the surface of an APC. The right panel (B) showsRTL302, a soluble single-chain molecule derived from the antigen-bindingif cell recognition domains. The structures are based on thecrystallographic coordinates of HLA-DR2 (PDB accession code 1BX2), andthe transmembrane domains are shown schematically as 0.5 nm cylinders.The amino and carboxyl termini of HLA-DR2 and RTL302 are labeled N, C,respectively. Disulfide bonds are displayed as ball and stick models.The lower right panel (C) shows the hydrophobic residues of thebeta-sheet platform of RTL302. Beta-sheet strands are depicted in ribbonform and the hydrophobic residues are grouped based on their locationwithin the beta-sheet platform and on their relative level ofinteraction with residues from the α2 and β2 Ig-fold domains. Group 1residues VI02, 1104, A106, F108, L110 comprised a central core alongbeta-strand 1 of the alpha-1 domain, and, peripheral to this core, L9and M119. Group II residues F19, L28, F32, V45, and V51 were beta-1domain residues and group III residues A133, V138 and L141 were from thealpha-1 domain.

We grouped these residues, based on their location within the beta-sheetplatform and on their relative level of interaction with residues fromthe α2 and β2 Ig-fold domains, and constructed a series of site-directedmutants, replacing single and then multiple residues with either serineor aspartic acid residues. The study developed in two stages, with thefirst stage focused on obtaining soluble proteins that weremonodisperse, and the second focused on biophysical and biochemicalcharacterization of the modified molecules. Reiterative site-directedmutagenesis allowed us to generate two modified RTLs that were suitablefor further biological characterization (TABLE I).

TABLE I Molecules used in this study Molecule Description RTL302 HumanHLA DR2 (DRB1*150101/DRA*0101) β1α1 domains RTL302 (5S) RTL302 (V102S,I104S, A106S, F108S, L110S)^(a) RTL302 (5D) RTL302 (V102D, I104D, A106D,F108D, L110D RTL303 RTL302/MBP-85-99^(b) RTL312 RTL302/MOG-35-55^(c)RTL320 RTL303 (5S) RTL340 RTL303 (5D) RTL342 RTL312 (5D) ^(a)RTL302numbering. These residues correspond to HLA-DR2 alpha-chain residues V6,I8, A10, F12, and L14. Residue numbering is increased in the Ag-tetheredmolecules to account for the Ag-peptide (variable length) plus linker(15 residues). ^(b)MBP-85-99, ENPVVHFFKNIVTPR ^(c)MOG-35-55,MEVGWYRSPFSRVVHLYRNGK

RTL302 could be converted to a monomer with either five serine (5S) orfive aspartate (5D) substitutions, RTL302(5S) and RTL302(5D),respectively, within a group of residues along the external face of thefirst strand of anti-parallel β-sheet derived from the alpha chain ofthe HLA-DR2 progenitor molecule. We have termed these the group I coreresidues (FIG. 1, left panel A). Comparison of the 5S or 5D modifiedmolecules with RTL302 by size exclusion chromatography (SEC) (FIG. 2,upper panel A) demonstrated that both RTL302(5S) and RTL302(5D) behavedas approximately 25 kD monomers.

FIG. 2 shows size exclusion chromatography of modified RTLs. Purifiedand refolded RTLs were analyzed by size exclusion chromatography (SEC).The upper panel (A) shows SEC of RTL302, RTL302(5S) and RTL302(5D).These RTLs do not contain covalently tethered Ag-peptides. The lowerpanel (B) shows SEC of RTLs derived from the wild-type HLA-DR2containing covalently tethered Ag-peptide MBP-85-99 (RTL303) orMOG-35-55 (RTL312). The 5S and 5D variants of RTL303 (RTL320, andRTL340, respectively) and the 5D variant of RTL312 (RTL342) are alsodisplayed. The Superdex 75 16/60 size exclusion column was calibratedwith a set of proteins of known molecular weight with exclusion volumesas indicated (*); Myoglobin, 17.3 kD; Ovalbumin, 43 kD; Bovine serumalbumin 67 kD; Catalase 232 kD; thyroglobulin, 670 kD.

Dynamic light scattering (DLS), was used to measure the diffusionconstants and calculate hydrodynamic radii for the molecules (Table II),and these studies demonstrated unequivocally that RTL302(5S) andRTL302(5D) were monomeric. When Ag-peptides were covalently tethered tothe amino-terminus of the molecules, their properties varied slightlydepending on the Ag-peptide used, and more importantly, differeddepending on the presence of the polar 5S or charged 5D modifications.Comparing RTL320 (5S modification, covalently tethered MBP-85-99peptide) with RTL340 (5D modification, covalently tethered MBP-85-99peptide), RTL320 still tended to aggregate, with most of the molecules(85%) formed into multimers of approximately 5 molecules. RTL340 wascompletely monomeric, and was more robust in terms of being able toaccommodate various covalently tethered Ag-peptides such as MBP-85-99(RTL340) and MOG-35-55 (RTL342) without significant alteration in thesolution properties of the RTLs (FIG. 2; Table II).

TABLE II Hydrodynamic analysis of RTLs by Dynamic Light ScatteringRadius Estimated % of Mass Molecule (nm) MW(kD) in buffer RTL302 (peakI)^(a) 17.6 2760 100 RTL302 (peak II) 2.5 27 100 RTL302 (5S) 2.5 27 98RTL302 (5D) 2.3 25 100 RTL303 15.4 2030 100 RTL312 (peak I) 15.2 1970 31RTL312 (peak II) 4.3 102 69 RTL320 (peak I) 13.5 1490 100 RTL320 (peakII) 4.8 131 100 RTL340 2.5 28 100 RTL342 2.6 31 100 Hydrodynamic Statusof modified RTLs were analyzed by light scattering analysis using aDynaPro ™ molecular sizing instrument (Protein Solutions, Inc.).^(a)Some of the proteins showed two clearly defined peaks by SEC andthese were characterized independently. Peak I refers to the aggregate(larger) peak, and peak II refers to the smaller size, in most casesmonomeric fraction.

Further biochemical analysis demonstrated that the 5S- and 5D-modifiedmolecules retained their native structure. RTLs contain a nativeconserved disulfide bond between cysteine 16 and 80 (RTL302 amino acidnumbering, corresponding to HLA-DR2 beta-chain residues 15 and 79). Airoxidation of these residues to reconstitute the native disulfide bondwas demonstrated by a gel shift assay in which identical samples with orwithout the reducing agent β-mercaptoethanol (β-ME) were boiled 5minutes prior to SDS-PAGE. In the absence of β-ME disulfide bonds areretained and proteins typically demonstrate a higher mobility duringelectrophoresis through acrylamide gels due to their more compactstructure. All of the RTL molecules produced showed this pattern,indicating the presence of the native conserved disulfide bond. Thesedata represent a primary confirmation of the conformational integrity ofthe molecules.

Circular dichroism (CD) demonstrated the highly ordered secondarystructures of the RTL constructs. The RTLs without covalently tetheredAg-peptide contained 20-25% alpha-helix, 21-27% anti-parallelbeta-strand, and 20-22% beta-turn structures (FIG. 3A; Table III).

TABLE III Secondary structure analysis of RTLs α- α-Parellel Parallel B-Random Molecule Helix β-Sheet β-Sheet turn coil Total RTL302 (peak I)0.21 0.21 0.02 0.23 0.33 0.99 RTL302 (peak II) 0.20 0.27 0.00 0.20 0.320.99 RTL302(5S) 0.20 0.21 0.02 0.22 0.34 1.00 RTL302(5D) 0.20 0.27 0.000.20 0.20 1.00 RTL303 0.26 0.20 0.04 0.19 0.32 1.00 RTL312 0.18 0.240.07 0.17 0.31 0.96 RTL320 (peak I) 0.22 0.22 0.03 0.21 0.32 1.00 RTL320(peak II) 0.19 0.19 0.03 0.23 0.35 1.00 RTL340 0.15 0.20 0.03 0.27 0.351.00 RTL342 0.19 0.22 0.05 0.18 0.30 0.93 Secondary structure contentderived from the deconvoluted spectra of the RTLs presented in FIG. 3.

FIG. 3 shows circular dichroism (CD) spectra of modified DR2-derivedRTLs. The upper panel (A) shows CD spectra of “empty” RTL302, RTL302(5S)and RTL302(5D). The middle panel (B) shows CD spectra of RTLs containingcovalently tethered Ag MBP-85-99 peptide. RTL303, RTL320, and RTL340.The lower panel (C) shows thermal denaturation curves for RTL303, RTL320and RTL340 show a high degree of cooperativity and stability. RTL340 wasresistant to complete thermal denaturation and aggregation and issoluble even after boiling for 5 minutes. Unless otherwise indicated, CDmeasurements were pedal arced at 25° C. on an Aviv-215 instrument using0.1 mm cell from 260 to 180nM on protein samples in 20 mM Tris-Cl, pH8.5. Concentration of each protein was determined by amino acidanalysis. Data are expressed as Delta-epsilon per mole per cm. Analysisof the secondary structure was performed using the variable selectionmethod (Compton et al., Analytical Biochemistry 155:155-67, 1986).

The RTLs with covalently tethered Ag-peptides contained 15-19%alpha-helix, 19-22% anti-parallel beta-strand, and 18-23% beta-turnstructures (FIG. 3B; Table III). These three basic secondary structuresof a polypeptide chain (helix, sheet, coil) each show a characteristicCD spectrum in the far UV, and a protein consisting of these elementsdisplays a spectra that can be deconvoluted into each of the individualcontributions. Although there are limitations inherent in the method(such as the lack of consideration of chromophore interaction(s) withindifferent structural regions), the fit is quite acceptable for whatwould be expected for a qualitative assessment of the RTL protein foldand is consistent with our previous data collected for the multimericversions of the RTLs (Chang et al., J. Biol. Chem 276:24170-76, 2001).The monodisperse monomeric RTLs retain the native structure of theprogenitor Ag-binding/TCR recognition domain of HLA-DR2.

We also used CD to monitor structure loss upon thermal denaturation. TheRTLs exhibited a high degree of thermal stability, and non-linearleast-square analysis indicated that the RTLs described in this studyare cooperatively folded (FIG. 3C). The temperature (T_(m)) at whichhalf of the structure was lost in 20 mM Tris, pH 8.5, was difficult todetermine because of the high melting temperatures observed.Extrapolation of the curves using non-linear analysis yields a T_(m), of92° C. for RTL303, 87° C. for RTL320 and 98° C. for RTL340. We hadpreviously reported a T_(m), for RTL303 of 78° C. when the molecule wassolubilized in PBS (Chang et al., J. Biol. Chem. 276:24170-76, 2001)reflecting the effect solvent had on the overall stability of themolecules.

We used a “peptide capture” ELISA assay with biotinylated--MOG tocompare Ag-peptide binding to RTL302, RTL302(5S), and RTL(5D).Non-linear regression analysis using a one-site (hyperbola) bindingmodel was used to calculate a B_(max) and K_(d) for the molecules (FIG.4A).

FIG. 4 shows direct measurement of peptide binding to HLA-DR2-derivedRTLs. Binding of biotinylated-MOG to RTL302 (open circles), RTL302(5S)(open diamonds), and RTL302(5D) (open squares). The left panel (A) showssaturation as a function of biotinylated-MOG concentration (insert showsScatchard analysis of peptide binding). The right panel (B) showsbinding of biotinylated-MOG peptide (0.15 μM) to RTLs as a function oftime to compare the initial rate of binding.

As shown in (FIG. 4B), binding of MOG peptide (0.15 μM) to RTLs as afunction of time was extremely fast. Using linear regression analysisthe initial rate of MOG binding was calculated to be 0.17±0.06 ΔOD/minfor RTL302, 0.11±0.02 ΔOD/min for RTL302(5S), and 0.10±0.02 for RTL(5D).

We characterized the in vitro activity of the RTLs in an assay designedto quantify their ability to induce Ag-specific inhibition of T cellproliferation (Burrows et al., J. Immunol. 167:4386-95, 2001; Wang etal., The Journal of Immunology, 2003; Vandenbark et al., Journal ofImmunology, 2003). The DR2-restricted T cell clone 4-G1 is specific forthe MBP-85-99 peptide. Cells that were not pretreated with RTLs(“untreated” control) showed a 68× stimulation index and cellspretreated with “empty” RTL302 showed close to 90× stimulation index, a31% increase above the “untreated” control. Pre-incubation with RTL303,RTL320 or RTL340 all showed greater than 90% inhibition of proliferationcompared with the “untreated” control (TABLE IV).

TABLE IV Antigen-specific inhibition of T cell proliferation bypre-incubating with RTLs Pre-incubation Clone EN4-G1 Untreated RTL302RTL303 RTL320 RTL340 +APC alone 588.97 569.1 578.7 592.0 641.9+APC/MBP85-99 40144.67 50841.1 2560.4 1847.7 1515.8 (10 μg/ml)Stimulation Index 68 89 4 3 2 Inhibition (%) — +31.1 −93.5 −95.2 −96.5Each data point represents the average of triple wells from eachtreatment.

We have recently described MOG-35-55-induced experimental autoimmuneencephalomyelitis (EAE) in DR2 (DRB1*1501) transgenic (Tg) mice(Vandenbark et al., Journal of Immunology, 2003). This animal model ofmultiple sclerosis (MS) was characterized as a moderately severe chronicdisease with 100% penetrance. Characteristics of the disease includeascending paralysis marked by inflammatory, demyelinating CNS lesions,EAE was induced with MOG-35-55 peptide/CFA on day 0 plus Ptx on days 0and 2, and the initial symptoms of disease could be observed beginningabout 10 days after induction. To evaluate the clinical potential of themonomeric RTL342, we treated Tg-DR2 mice with MOG-induced EAE 2-4 daysafter onset of clinical signs with RTL312, RTL342, or vehicle alone(FIG. 5, and see also TABLE V).

FIG. 5 shows that monomeric, monodisperse RTL342 was as effective asRTL312 at treating EAE in DR*1501transgenic animals. Mean clinicalscores of HLA DR2 (DRB1*1501/DRA*0101) transgenic mice treated with 33μg of RTL312 (v), RTL342 (Δ), or vehicle alone (Tris, pH 8.5) (). Allmice were immunized s.c. with 200 μg MOG-35-55 and 400 μg CF A inconjunction with 100 ng Ptx i.v, on Day 0 and 266 ng Ptx 2 dayspost-immunization. On Day 14 all mice were distributed into 6 groupsaccording to similarity in disease and gender. Mice were i.v. injecteddaily with RTL312, RTL342, or vehicle. (n=4 per group, except forvehicle group where n=3; arrows indicate treatment).

Treatment with RTL312 or RTL342 rapidly reversed established clinicalsigns of EAE (score about 2.5) to an average daily score of <0.5 unitsby the end of the eight-day treatment period. This low degree ofdisability was maintained without further RTL injections over theremainder of the observation period, which in one experiment lasted for5 weeks after treatment was stopped. In contrast to the reversal of EAEmediated by RTL312 or RTL342, control groups receiving vehicle or 33μg/injection of non-Ag-specified RTL303 (containing MBP-85-99 peptide)developed moderately severe chronic EAE (score of >4),

TABLE V RTL treatment of DR2 Transgenic Mice Inci- Mor- Treatment denceOnset Peak tality CDI RTL312 4/4  9.5 ± 2.8   3 ± 1.8 0/4 29.8 ± 21.7*RTL342 4/4 10.8 ± 2.2 2.6 ± 1.1 0/4  16 ± 10.9* Vehicle 3/3 12.3 ± 1.2 6± 0 0/4 133.7 ± 11.1  *= Significant difference between experimentalgroup and vehicle group (p = 0.000)

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by persons of ordinaryskill in the art to which this invention pertains.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the methodsand methodologies that are described in the publications which may beused in connection with the presently described invention. Thepublications discussed above and throughout the text are provided solelyfor their public disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior invention.

It will be understood by those skilled in the art that the foregoingdescription is intended to illustrate and not limit the scope of theinvention defined in part by the appended claims and otherwise supportedby the disclosure herein. Other aspects, advantages, and modificationsof the current invention will be appreciated as embodied within thescope of the present disclosure, including but not limited tocompositions, methods, devices and kits employing substantially similaror equivalent subject matter as described herein, which will berecognized as useful for practicing the invention and for implementingadditional or alternative refinements, improvements, or relatedapplications thereof.

What is claimed is:
 1. An isolated, modified recombinant T cell ligand(RTL) having a reduced potential for aggregation in solution,comprising: a major histocompatibility complex (MHC) component in theform of a single chain (sc) polypeptide comprising multiple,covalently-linked MHC domain elements selected from a) α1 and β1 domainsof an MHC class II polypeptide, or portions thereof comprising anAg-binding pocket/T cell receptor (TCR) interface; or b) α1 and α2domains of an MHC class I polypeptide, or portions thereof comprising anAg-binding pocket/TCR interface, wherein the MHC component is modifiedby one or more amino acid substitution(s), addition(s), deletion(s), orrearrangement(s) at a target site corresponding to a self-associatinginterface identified in a native MHC polypeptide or RTL comprising thenative MHC polypeptide, whereby the modified RTL exhibits reducedaggregation in solution compared to aggregation exhibited by anunmodified, control RTL having the MHC component structure set forth ina) or b) but incorporating the native MHC polypeptide having an intactself-associating interface.
 2. The isolated, modified RTL of claim 1,wherein the modified RTL comprises an MHC class II MHC component.
 3. Theisolated, modified RTL of claim 2, wherein the MHC class II MHCcomponent comprises α1 and β1 domains of an HLA-DR protein, or portionsthereof comprising an Ag-binding pocket/T cell receptor (TCR) interface.4. The isolated, modified RTL of claim 2, wherein the MHC class II MHCcomponent comprises α1 and β1 domains of an HLA-DQ protein, or portionsthereof comprising an Ag-binding pocket/T cell receptor (TCR) interface.5. The isolated, modified RTL of claim 2, wherein the MHC class II MHCcomponent comprises α1 and β1 domains of an HLA-DP protein, or portionsthereof comprising an Ag-binding pocket/T cell receptor (TCR) interface.6. The isolated, modified RTL of claim 2, wherein the MHC class H MHCcomponent excludes all or part(s) of α2 and β2 domains of acorresponding, native MHC class II molecule.
 7. The isolated, modifiedRTL of claim 6, wherein the MHC class II MHC component excludes a CD4interactive domain of the corresponding, native MHC class H molecule. 8.The isolated, modified RTL of claim 2, wherein the MHC class II MHCcomponent comprises α1 and β1 domains of a mammalian MHC class IImolecule, wherein an amino terminus of the α1 domain is covalentlylinked to the carboxy terminus of the β1 domain to form a single chain(sc) MHC component.
 9. The isolated, modified RTL of claim 8, whereinthe α1 and 62 1 domains are coupled by a peptide linker.
 10. Theisolated, modified RTL of claim 1, further comprising a T cell antigenicdeterminant bound to the Ag-binding pocket of the MHC component orcovalently linked to the MHC component.
 11. The isolated, modified RTLof claim 1, combined with or coupled to a toxin effective to mediate Tcell killing.
 12. The isolated, modified RTL of claim 1, wherein the RTLis modified by one or more amino acid substitution(s) or deletion(s) atone or more target site(s) characterized by the presence of ahydrophobic residue within a β-sheet platform of a native MHCpolypeptide or RTL comprising the native MHC polypeptide.
 13. Theisolated, modified RTL of claim 12, wherein said one or more targetsites define a self-binding motif within β-sheet platform central coreof the native MHC polypeptide or RTL comprising the native MHCpolypeptide.
 14. The isolated, modified RTL of claim 13, wherein saidone or more target sites comprise(s) one or any combination of residuesof the central core portion of the β-sheet platform selected from V102,I104, A106, F108, and L110.
 15. The isolated, modified RTL of claim 14,wherein said one or combination of residues is/are modified bysubstitution with a non-hydrophobic amino acid.
 16. The isolated,modified RTL of claim 15, wherein said one or combination of residuesis/are modified by substitution with a polar or charged amino acid. 17.The isolated, modified RTL of claim 15, wherein said one or combinationof residues is/are modified by substitution with a serine or aspartateresidue.
 18. The isolated, modified RTL of claim 15, wherein said one orcombination of residues is/are modified by substitution with a serine oraspartate residue.
 19. The isolated, modified RTL of claim 14, whereineach of the residues V102, I104, A106, F108, and L110 of the centralcore portion of the β-sheet are modified by substitution with anon-hydrophobic amino acid.
 20. The isolated, modified RTL of claim 13,wherein said one or more target sites comprise(s) one or any combinationof residues of the β-sheet platform selected from L9, F19, L28, F32,V45, V51, A133, V138, and L141.
 21. The modified RTL of claim 10,wherein the modified RTL bound or linked to the Ag— is effective tomodulate T cell activity in a T cell receptor (TCR)-mediated,Ag-specific manner.
 22. The modified RTL of claim 21, wherein themodified RTL bound or linked to the Ag— is effective to inhibit T cellproliferation or inflammatory cytokine production in vitro or in vivo.23. The modified RTL of claim 22, wherein the modified RTL bound orlinked to the Ag— is effective to reduce a pathogenic activity orpathogenic potential of a T cell associated with an autoimmune diseasein a mammalian cell or subject.
 24. An assay composition or kit usefulto detect, quantify and/or purify antigen-specific T-cells, comprising amodified, recombinant T cell receptor ligand (RTL) according to claim 1or claim
 2. 25. A composition useful to modulate T cell phenotype,activity, differentiation status, migration behavior, tissuelocalization, and/or cellular fate, comprising a modified, recombinant Tcell receptor ligand (RTL) according to claim 1 or claim
 2. 26. Thecomposition of claim 25, which is effective for modulating T cellactivation, proliferation, and/or expression of one or more cytokine(s),growth factor(s), chemokines, cell surface receptors, and/or cellularadhesion molecules (CAMs).
 27. A pharmaceutical formulation fortreatment of an autoimmune disease or disorder in a mammalian subjectcomprising a modified, recombinant T cell receptor ligand (RTL)according to claim 1 or claim 2.